Cell lines with latent immunodeficiency virus and methods of use thereof

ABSTRACT

The present invention provides isolated cells that comprise, integrated into the genome of the cell, a transcription-competent immunodeficiency virus or a transcription-competent immunodeficiency virus-based retroviral vector. Under basal in vitro culture conditions, the immunodeficiency virus is latent, and the expression of the latent immunodeficiency virus can be reactivated. The invention further provides methods of making a subject cell. The invention further provides screening methods for identifying agents that activate a latent immunodeficiency virus; and screening method for identifying agents that block reactivation of latent immunodeficiency virus expression in response to T cell activation signals. The invention further provides agents identified in the subject screening assays. The invention further provides methods of treating an immunodeficiency virus infection.

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/341,727 filed Dec. 19, 2001, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant nos.ROI-GM51671-05A2 awarded by the National Institutes of Health. Thegovernment may have certain rights in this invention.

FIELD OF THE INVENTION

[0003] This invention is in the field of immunodeficiency viruses,particularly recombinant immunodeficiency viruses, and cell linescontaining same.

BACKGROUND OF THE INVENTION

[0004] Combination antiretroviral therapy can control HIV-1 replicationand delay disease progression. However, despite the complete suppressionof detectable viremia in many patients, viremia reemerges rapidly afterinterruption of treatment, consistent with the existence of a latentviral reservoir. This reservoir is thought to consist mainly of latentlyinfected resting memory CD4⁺ T cells. Due to the long half-life of thisreservoir (44 months), it has been estimated that its total eradicationwith current treatment would require over 60 years.

[0005] Latently infected cells contain replication-competent integratedHIV-1 genomes that are blocked at the transcriptional level, resultingin the absence of viral protein expression. HIV depends on both cellularand viral factors for efficient transcription of its genome, and theactivity of the HIV promoter is tightly linked to the level ofactivation of its host cell. It is thought that reactivation of latentlyinfected memory T cells by their cognate antigen leads to a reactivationof viral gene expression and the completion of the viral life cycle.However, it is not clear how the latent state is established. It hasbeen proposed that latency occurs when an activated T cell in the earlystage of infection returns to a quiescent state, leading to suppressionof viral transcription until the cell becomes reactivated. However, theinfected cell would need to survive the cytopathic effects of infectionto effectively transition to the resting state. Alternatively, anactivated, infected cell could become quiescent before the onset ofviral expression and the occurrence of cytopathic effects, as has beenreported during thymopoiesis. The unlikely coincidence of these twoevents could account for the low frequency of latently infected cells invivo (˜10⁶ cells per infected individual).

[0006] HIV transcription is characterized by two temporally distinctphases. The early phase occurs immediately after integration and reliessolely on cellular transcription factors. Because of a transcriptionalelongation defect in the basal HIV promoter, most transcripts cannotelongate efficiently and terminate rapidly after initiation. Thisprocess leads to the accumulation of short transcripts at the 5′ regionof the viral genome containing the TAR element. However, the elongationdefect is not absolute, and a few transcripts elongate throughout thegenome, resulting in transcription of the viral transactivator Tat. Thelate phase of transcription occurs when enough Tat protein hasaccumulated. Tat binds to TAR, recruits the pTEFb complex, and causesthe hyperphosphorylation of RNA polymerase II, dramatically increasingits ability to elongate.

[0007] To understand how postintegration latency is established and totest novel therapeutic approaches for the reactivation of these viralreservoirs, an in vitro cell system reflecting the state of HIV-1latency is required. Several HIV latently infected cell lines harbordefective proviruses, raising significant questions about theirsignificance in understanding the mechanism of latency in vivo. Thelatent cell lines ACH2 (T cell) and U1 (promonocytic) contain HIVproviruses that harbor mutations in their Tat-TAR transcriptional axis.Another chronically infected cell line, J-delta-k (T-cell), harbors anHIV-1 provirus lacking NF-κB binding sites in the HIV promoter. Theseobservations suggest that inhibition of transcription is critical toestablishment and maintenance of latency.

[0008] There is a need in the art for an in vitro cell system thataccurately reflects latent immunodeficiency virus infection in vivo. Thepresent invention addresses this need.

[0009] Literature

[0010] Kulkosky et al. (2001) Blood 98:3006-3015; Emiliani et al. (1996)Proc. Natl. Acad. Sci. USA 93:6377-6381; Emiliani et al. (1998) J.Virol. 72:1666-1670; Antoni et al. (1994) Virol. 202:684-694; Carteau etal. (1998) J. Virol. 72:4005-4014; U.S. Pat. Nos. 6,225,048; 6,025,124;5,459,056; and 5,256,534. See U.S. Pat. No. 6,025,124 for a discussionof U1 cells; see U.S. Pat. No. 5,459,056 for a discussion of ACH-2cells. See U.S. Pat. No.5,256,534 for a discussion of OM-10.1 cells.

SUMMARY OF THE INVENTION

[0011] The present invention provides isolated cells that comprise,integrated into the genome of the cell, a transcription-competentimmunodeficiency virus or a transcription-competent immunodeficiencyvirus-based retroviral vector. Under basal in vitro culture conditions,the immunodeficiency virus is latent, and the expression of the latentimmunodeficiency virus can be reactivated. The invention furtherprovides methods of making a subject cell. The invention furtherprovides screening methods for identifying agents that activate a latentimmunodeficiency virus; and screening method for identifying agents thatblock reactivation of latent immunodeficiency virus expression inresponse to T cell activation signals. The invention further providesagents identified in the subject screening assays. The invention furtherprovides methods of treating an immunodeficiency virus infection.

FEATURES OF THE INVENTION

[0012] The present invention features an isolated cell that comprises,integrated into the genome of the cell, a recombinanttranscription-competent immunodeficiency virus or virus-based vector,wherein, under basal in vitro culture conditions, the immunodeficiencyvirus is latent, and wherein expression of the latent immunodeficiencyvirus can be reactivated. In many embodiments, the isolated cell is animmortalized cell line. In some embodiments, the isolated cell is a Tlymphoid cell.

[0013] In many embodiments, the immunodeficiency virus is humanimmunodeficiency virus (HIV), e.g., HIV-1.

[0014] The invention further features a method of making an immortalizedcell that comprises, integrated into the genome of the cell, arecombinant, transcription-competent recombinant human immunodeficiencyvirus (HIV), wherein, under basal in vitro culture conditions, the HIVis latent, and wherein expression of the latent HIV can be reactivated.The method generally involves introducing into population ofimmortalized cells in vitro a recombinant, transcription-competent HIVthat comprises a nucleotide sequence encoding a selectable markeroperably linked to a promoter; and selecting a cell population thatcomprises the recombinant HIV integrated into the genome of the cell,and that does not produce the detectable marker. In many embodiments,the method further comprises cloning a cell from the selected cellpopulation. In many embodiments, the selection step results in a firstselected cell population, and the method further comprises the steps ofcontacting the first selected cell population with an agent thatactivates HIV transcription; and selecting a second population of cellsfrom the first selected population, which second selected populationproduces the selectable marker. Activating agents include, e.g., anactivator of NF-κB, an agent that cross-links cell-surface T-cellreceptor, and an inhibitor of histone deacetylase. Non-limiting examplesof activating agents are phytohemagglutinin, tetradecanoyl phorbolacetate , TNFα, an anti-CD3 antibody, and trichostatin A.

[0015] The invention further features an isolated immortalized cell thatcomprises, integrated into the genome of the cell, a recombinanttranscription-competent human immunodeficiency virus (HIV) thatcomprises a nucleotide sequence encoding a selectable marker operablylinked to a promoter, wherein, under basal in vitro culture conditions,the HIV is latent, and wherein expression of the latent HIV can bereactivated.

[0016] The invention further features a method of identifying an agentthat activates a latent human immunodeficiency virus (HIV). The methodgenerally involves contacting a cell with a test agent, which cell is anisolated immortalized cell that comprises, integrated into the genome ofthe cell, a recombinant transcription-competent human immunodeficiencyvirus (HIV) that comprises a nucleotide sequence encoding a selectablemarker operably linked to a promoter, wherein, under basal in vitroculture conditions, the HIV is latent, and wherein expression of thelatent HIV can be reactivated; and determining the effect, if any, ofthe test agent on production of the detectable marker, whereinproduction of the detectable marker indicates that the test agentactivates a latent HIV. In many embodiments, the detectable marker is afluorescent protein, and the determining step is detection offluorescence.

[0017] The invention further features an active agent that reactivateslatent immunodeficiency virus, which agent is identified by a screeningmethod of the invention, and compositions comprising the active agent.In many embodiments, the active agent is formulated in a compositionwith a pharmaceutically acceptable excipient.

[0018] The invention further features a method of reducing the number ofcells containing a latent human immunodeficiency virus in an individual.The method generally involves administering to the individual aneffective amount of a composition comprising a subject active agent.Generally, the active agent is administered as part of a combinationtherapy with an anti-HIV therapeutic agent.

[0019] The invention further features a method of treating a humanimmunodeficiency virus infection in an individual. The method generallyinvolves administering to an individual an effective amount of acomposition comprising a subject active agent; and administering to theindividual an effective amount of an agent that inhibits animmunodeficiency virus function selected from the group consisting ofviral replication, viral protease activity, viral reverse transcriptaseactivity, viral entry into a cell, viral integrase activity, viral Revactivity, viral Tat activity, viral Nef activity, viral Vpr activity,viral Vpu activity, and viral Vif activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGS. 1A-C depict enrichment and purification of HIV-latentlyinfected cells by FACS.

[0021] FIGS. 2A-C depict characterization of latently infected clones.

[0022] FIGS. 3A-D depict data showing that latency is associated withpreferred integration in or near alphoid repeats.

[0023]FIG. 4 depicts transcriptional activation of the HIV promoter inlatently infected cells.

[0024] FIGS. 5A-C depict establishment of latently infected cell lineswith a full-length HIV provirus.

[0025]FIG. 6 depicts the nucleotide sequences at the integration site ofprovirus integrated into alphoid repeats from PBMCs from HIV-1-infectedindividuals treated with highly active antiretroviral therapy.

[0026]FIG. 7 depicts reactivation of latent HIV by two different agents.

DEFINITIONS

[0027] As used herein, the terms “treatment”, “treating”, and the like,refer to obtaining a desired pharmacologic and/or physiologic effect.The effect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, e.g., causing regression of the disease,e.g., to completely or partially remove symptoms of the disease. In thecontext of immunodeficiency virus infection, the term “treatment”encompasses prevention of establishment of a systemic infectionfollowing initial contact with the virus; and prophylactic treatment ofan individual not yet infected with the virus.

[0028] The terms “individual,” “host,” “subject,” and “patient,” usedinterchangeably herein, refer to a mammal, including, but not limitedto, murines, felines, simians, humans, mammalian farm animals, mammaliansport animals, and mammalian pets. The term includes mammals that aresusceptible to infection by an immunodeficiency virus.

[0029] A “biological sample” encompasses a variety of sample typesobtained from an individual and can be used in a diagnostic ormonitoring assay. The definition encompasses blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived therefrom and the progenythereof. The definition also includes samples that have been manipulatedin any way after their procurement, such as by treatment with reagents;washed; or enrichment for certain cell populations, such as CD4⁺ Tlymphocytes, glial cells, macrophages, tumor cells, peripheral bloodmononuclear cells (PBMC), and the like. The term “biological sample”encompasses a clinical sample, and also includes cells in culture, cellsupernatants, tissue samples, organs, bone marrow, and the like.

[0030] The term “immunodeficiency virus” as used herein, refers to humanimmunodeficiency virus-1 (HIV-1); human immunodeficiency virus-2(HIV-2); any of a variety of HIV subtypes and quasispecies; simianimmunodeficiency virus (SIV); and feline immunodeficiency virus (FIV).As used herein in the context of latent immunodeficiency virus in asubject isolated cell, the term also includes immunodeficiencyvirus-based retroviral vectors (e.g., recombinant immunodeficiencyvirus).

[0031] The term “transcription competent” as used herein in the contextof transcription-competent latent immunodeficiency virus, refers to alatent immunodeficiency virus (including latent immunodeficiencyvirus-based retroviral vectors) that encodes functional Tat and has afunctional TAR site in the LTR.

[0032] The term “latent,” as used herein in the context of a latentimmunodeficiency virus refers to a genomically integratedimmunodeficiency virus (including a latent immunodeficiency virus-basedretroviral vector, e.g., a recombinant immunodeficiency virus) that istranscriptionally silent, e.g., immunodeficiency virus transcripts areundetectable or are at background levels, in a cell comprising thelatent immunodeficiency virus.

[0033] The term “reactivated,” as used herein in the context of in vivoreactivated immunodeficiency virus, refers to an immunodeficiency virusthat, after a period of latency, becomes transcriptionally active, andin many instances forms infectious viral particles. The term“reactivated,” as used herein in the context of in vitro reactivatedimmunodeficiency virus in a subject cell, refers to an immunodeficiencyvirus (e.g., a recombinant immunodeficiency virus) that, after a periodof latency, becomes transcriptionally active, i.e., a functional Tatprotein mediates transcription from a functional immunodeficiency viruspromoter (e.g., a long terminal repeat promoter). In those embodimentsin which a reactivated recombinant immunodeficiency virus is notreplication competent, the recombinant immunodeficiency virus can bepackaged into infectious particles by providing any missing viralproteins (e.g., gag and env proteins) via a helper virus.

[0034] As used herein the term “isolated,” in the context of a subjectisolated cell, refers to a cell that is in an environment different fromthat in which the cell naturally occurs. As used herein, the term“clonal cell line” refers to a cloned cell line that is typicallyimmortalized, e.g., under suitable in vitro culture conditions, the cellline divides virtually indefinitely. Isolated cells are also referred toherein as “host cells.”

[0035] The term “host cell” includes an individual cell or cell culturewhich can be or has been a recipient of any recombinant vector(s) orconstruct of the invention. Host cells include progeny of a single hostcell, and the progeny may not necessarily be completely identical (inmorphology or in total DNA complement) to the original parent cell dueto natural, accidental, or deliberate mutation and/or change. A hostcell includes cells tranfected or infected in vitro with a recombinantvector or a construct of the invention. A host cell which comprises arecombinant vector or construct of the invention is a “recombinant hostcell.”

[0036] “Alkyl” is a monovalent, saturated or unsaturated, straight,branched or cyclic, aliphatic (i.e., not aromatic) hydrocarbon group. Invarious embodiments, the alkyl group has 1-20 carbon atoms, i.e., is aC1-C20 (or C₁-C₂₀) group, or is a C1-C18 group, a C1-C12 group, a C1-C6group, or a C1-C4 group. Independently, in various embodiments, thealkyl group: has zero branches (i.e., is a straight chain), one branch,two branches, or more than two branches; is saturated; is unsaturated(where an unsaturated alkyl group may have one double bond, two doublebonds, more than two double bonds, and/or one triple bond, two triplebonds, or more than three triple bonds); is, or includes, a cyclicstructure; is acyclic. Exemplary alkyl groups include C₁alkyl (i.e.,—CH₃ (methyl)), C₂alkyl (i.e., —CH₂CH₃ (ethyl), —CH═CH₂ (ethenyl) and—C≡CH (ethynyl)) and C₃alkyl (i.e., —CH₂CH₂CH₃ (n-propyl), —CH(CH₃)₂(i-propyl), —CH═CH—CH₃ (1-propenyl), —C≡C—CH₃ (1-propynyl), —CH₂—CH═CH₂(2-propenyl),—CH₂—C≡CH (2-propynyl), —C(CH₃)═CH₂ (1-methylethenyl), and—CH(CH₂)₂ (cyclopropyl)).

[0037] “Ar” indicates a carbocyclic aryl group selected from phenyl,substituted phenyl, naphthyl, and substituted naphthyl. Suitablesubstituents on a phenyl or naphthyl ring include C₁-C₆alkyl,C₁-C₆alkoxy, carboxyl, carbonyl(C₁-C₆)alkoxy, halogen, hydroxyl, nitro,—SO₃H, and amino.

[0038] “Aryl” is a monovalent, aromatic, hydrocarbon, ring system. Thering system may be monocyclic or fused polycyclic (e.g., bicyclic,tricyclic, etc.). In various embodiments, the monocyclic aryl ring isC5-C10, or C5-C7, or C5-C6, where these carbon numbers refer to thenumber of carbon atoms that form the ring system. A C6 ring system,i.e., a phenyl ring, is a preferred aryl group. In various embodiments,the polycyclic ring is a bicyclic aryl group, where preferred bicyclicaryl groups are C8-C12, or C9-C10.

[0039] “Arylene” is a polyvalent, aromatic hydrocarbon, ring system. Thering system may be monocyclic or fused polycyclic (e.g., bicyclic,tricyclic, etc.). In some embodiments, the monocyclic arylene group isC5-C10, or C5-C7, or C5-C6, where these carbon numbers refer to thenumber of carbon atoms that form the ring system. A C6 ring system,i.e., a phenylene ring, is an exemplary aryl group. In some embodiments,the polycyclic ring is a bicyclic arylene group, where exemplarybicyclic arylene groups are C8-C12, or C9-C10. The arylene group may bedivalent, i.e., it has two open sites that each bond to another group;or trivalent, i.e., it has three open sites that each bond to anothergroup; or it may have more than three open sites.

[0040] “Carbocycle” refers to a ring formed exclusively from carbon,which may be saturated or unsaturated, including aromatic. The ring maybe monocyclic (e.g., cyclohexyl, phenyl), bicyclic (e.g., norbornyl),polycyclic (e.g., adamantyl) or contain a fused ring system (e.g.,decalinyl, naphthyl). In one embodiment, the ring is monocyclic andformed from 5, 6 or 7 carbons. In one embodiment, the ring is bicyclicand formed from 7, 8 or 9 carbons. In one embodiment, the ring ispolycyclic and formed from 9, 10 or 11 carbons. In one embodiment, thering includes a fused ring system and is formed from 8-12 carbons. Thus,in one embodiment, the carbocycle is formed from 5-12 ring carbons.

[0041] “Heteroalkyl” is an alkyl group (as defined herein) wherein atleast one of the carbon atoms is replaced with a heteroatom. Exemplaryheteroatoms are nitrogen, oxygen, sulfur, and halogen. A heteroatom may,but typically does not, have the same number of valence sites as carbon.Accordingly, when a carbon is replaced with a heteroatom, the number ofhydrogens bonded to the heteroatom may need to be increased or decreasedto match the number of valence sites of the heteroatom. For instance, ifcarbon (valence of four) is replaced with nitrogen (valence of three),then one of the hydrogens formerly attached to the replaced carbon mustbe deleted. Likewise, if carbon is replaced with halogen (valence ofone), then three (i.e., all) of the hydrogens formerly bonded to thereplaced carbon must be deleted. As another example, trifluoromethyl isa heteroalkyl group wherein the three methyl groups of a t-butyl groupare replaced by fluorine.

[0042] “Heteroalkylene” is an alkylene group (as defined herein) whereinat least one of the carbon atoms is replaced with a heteroatom.Exemplary heteroatoms are nitrogen, oxygen, sulfur, and halogen. Aheteroatom may, but typically does not, have the same number of valencesites as carbon. Accordingly, when a carbon is replaced with aheteroatom, the number of hydrogens bonded to the heteroatom may need tobe increased or decreased to match the number of valence sites of theheteroatom, as explained elsewhere herein.

[0043] “Heteroaryl” is a monovalent aromatic ring system containingcarbon and at least one heteroatom in the ring. The heteroaryl groupmay, in various embodiments, have one heteroatom, or 1-2 heteroatoms, or1-3 heteroatoms, or 1-4 heteroatoms in the ring. Heteroaryl rings may bemonocyclic or polycyclic, where the polycyclic ring may contained fused,spiro or bridged ring junctions. In one embodiment, the heteroaryl isselected from monocyclic and bicyclic. Monocyclic heteroaryl rings maycontain from about 5 to about 10 member atoms (carbon and heteroatoms),e.g., from 5-7, and most often from 5-6 member atoms in the ring.Bicyclic heteroaryl rings may contain from about 8-12 member atoms, or9-10 member atoms in the ring. The heteroaryl ring may be unsubstitutedor substituted. In one embodiment, the heteroaryl ring is unsubstituted.In another embodiment, the heteroaryl ring is substituted. Exemplaryheteroaryl groups include benzofuran, benzothiophene, furan, imidazole,indole, isothiazole, oxazole, piperazine, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, quinoline, thiazole andthiophene.

[0044] “Heteroarylene” is a polyvalent aromatic ring system containingcarbon and at least one heteroatom in the ring. In other words, aheteroarylene group is a heteroaryl group that has more than one opensite for bonding to other groups. The heteroarylene group may, invarious embodiments, have one heteroatom, or 1-2 heteroatoms, or 1-3heteroatoms, or 1-4 heteroatoms in the ring. Heteroarylene rings may bemonocyclic or polycyclic, where the polycyclic ring may contained fused,spiro or bridged ring junctions. In one embodiment, the heteroaryl isselected from monocyclic and bicyclic. Monocyclic heteroarylene ringsmay contain from about 5 to about 10 member atoms (carbon andheteroatoms), preferably from 5-7, and most preferably from 5-6 memberatoms in the ring. Bicyclic heteroarylene rings may contain from about8-12 member atoms, or 9-10 member atoms in the ring.

[0045] “Heteroatom” is a halogen, nitrogen, oxygen, silicon or sulfuratom. Groups containing more than one heteroatom may contain differentheteroatoms.

[0046] “Heterocycle” refers to a ring containing at least one carbon andat least one heteroatom. The ring may be monocyclic (e.g., morpholinyl,pyridyl), bicyclic (e.g., bicyclo[2.2.2]octyl with a nitrogen at onebridgehead position), polycyclic, or contain a fused ring system. In oneembodiment, the ring is monocyclic and formed from 5, 6 or 7 atoms. Inone embodiment, the ring is bicyclic and formed from 7, 8 or 9 atoms. Inone embodiment, the ring is polycyclic and formed from 9, 10 or 11atoms. In one embodiment, the ring includes a fused ring system and isformed from 8-12 atoms. Thus, in one embodiment, the heterocycle isformed from 5-12 ring atoms. In one embodiment, the heteroatom isselected from oxygen, nitrogen and sulfur. In one embodiment, theheterocycle contains 1, 2 or 3 heteroatoms.

[0047] “Pharmaceutically acceptable salt” and “salts thereof” in thecompounds of the resent invention refers to acid addition salts and baseaddition salts.

[0048] Acid addition salts refer to those salts formed from compounds ofthe present invention and inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and/or organic acids such as acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

[0049] Base addition salts refer to those salts formed from compounds ofthe present invention and inorganic bases such as sodium, potassium,lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese,aluminum salts and the like. Suitable salts include the ammonium,potassium, sodium, calcium and magnesium salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaines, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, and the like.

[0050] Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

[0051] Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

[0052] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

[0053] It must be noted that as used herein and in the appended claims,the singular forms “a”, “and”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a cell” includes a plurality of such cells and reference to “the agent”includes reference to one or more agents and equivalents thereof knownto those skilled in the art, and so forth.

[0054] The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention provides an isolated cell that comprises,integrated into the genome of the cell, a transcription-competentimmunodeficiency virus or a transcription-competent immunodeficiencyvirus-based retroviral vector. Under basal in vitro culture conditions,the immunodeficiency virus is latent, and the expression of the latentimmunodeficiency virus can be reactivated. The latent immunodeficiencyvirus is transcriptionally inactive, but is fully transcriptionallycompetent. Previously available cell lines such as ACH2, U1, A2.01, andderivatives of such cell lines, contain latent HIV that is nottranscriptionally competent. The latent HIV in such cell lines containsmutations in Tat and/or in the LTR, e.g., in the TAR region or the NFκBbinding site. Therefore, the previously available cell lines are notrepresentative of the cells in a reservoir of latently infected cells inan infected individual. The isolated cells of the instant invention aretranscriptionally competent, e.g., the Tat protein is functional, andthe viral LTR is functional (e.g., has functional TAR and other elementsnecessary for transcriptional activation. Thus, the subject cells arerepresentative of cells in a reservoir of latently infected cells in aninfected individual.

[0056] The subject cells are useful in screening methods for identifyingagents that activate latent immunodeficiency virus. Agents that activatelatent immunodeficiency virus are useful, particularly in combinationwith established anti-HIV therapeutic agents, to reduce the reservoir oflatently infected cells in an HIV-infected individual. Such agents areuseful to reduce or eliminate the problem of reemergence of viremiafollowing cessation or interruption of treatment with anti-HIVtherapeutic agent(s), and therefore render existing therapeutic agentsmore effective.

[0057] The subject cells are also useful in screening methods foridentifying agents that block or reduce reactivation of latentimmunodeficiency virus transcription in response to T cell activationsignals. Such agents are useful to suppress reactivation of a latentimmunodeficiency virus.

[0058] The invention further provides methods of making a subject cell(e.g., a cell that harbors a latent, transcription competentimmunodeficiency virus). The invention further provides screeningmethods for identifying agents that activate a latent immunodeficiencyvirus; and screening methods for identifying agents that suppress orblock activation of a latent immunodeficiency virus. The inventionfurther provides agents identified using a screening method of theinvention. The invention further provides methods of treating an HIVinfection, with the-goal of eradicating the HIV from the infectedindividual (in contrast to suppressing the infection, as is achievedusing currently applied anti-HIV therapy).

[0059] Isolated Cells with Latent Immunodeficiency Virus

[0060] The present invention provides an isolated cell that comprises,integrated into the genome of the cell, a transcription-competentimmunodeficiency virus or a transcription-competent immunodeficiencyvirus-based retroviral vector. While human immunodeficiency virus (HIV)is exemplified in this specification, the disclosure pertains to otherimmunodeficiency viruses as well and is not meant to be limited to HIV.

[0061] Under basal in vitro culture conditions, the genomicallyintegrated HIV is latent, and the expression of the latent HIV can bereactivated. The subject cells are useful as in vitro models for alatent HIV infection in vivo and for screening for agents that activatelatent HIV.

[0062] The latent HIV is transcriptionally inactive under basal in vitroculture conditions, but is fully transcriptionally competent. “Basal invitro culture conditions” typically involve standard culture media, atemperature of about 37° C., and 5% CO₂. Standard culture media include,but are not limited to, RPMI 1640 medium, McCoy's 5A medium, Leibovitz'sL15 medium, Eagle's minimal essential medium, Dulbecco's modifiedEagle's medium, and the like. In many cases, the medium will besupplemented with additional components, e.g., 10 mM HEPES buffer; 2 mML-glutamine; 100 U/ml penicillin; 100 μg/ml streptomycin; andheat-inactivated fetal calf serum, in an amount (in volume/volume) offrom about 5% to about 10%, from about 10% to about 15%, or from about15% to about 20%, or higher.

[0063] Basal in vitro culture conditions generally exclude the presencein the medium of a factor(s) that would activate HIV transcriptionand/or production of HIV virions, including the factors listed below forreactivation of latent HIV.

[0064] Under certain cell culture conditions, the latent HIV can bereactivated, e.g., the latent HIV becomes transcriptionally activated.In many embodiments, reactivation of the latent virus (or recombinantvirus) does not require a host cell factor). Culture conditions thatresult in reactivation of latent HIV include contacting the cell for asuitable period of time with an effective amount of one or morereactivating agents. Agents that reactivate latent HIV in a subject cellare termed “reactivating agents” and include, but are not limited to,activators of NF-κB, including, but not limited to, phytohemagglutinin(PHA), phorbol esters, e.g., tetradecanoyl phorbol acetate (TPA), andTNFα; exposure to an antigen for which a cell surface T-cell receptor isspecific; an agent that cross-links cell-surface T-cell receptor, e.g.,anti-CD3 antibody; inhibitors of histone deacetylase, e.g., trichostatinA, sodium butyrate, and trapoxin.

[0065] An effective amount of a reactivating agent is an amounteffective to achieve transcriptional activation of the latentimmunodeficiency virus. Whether transcription is reactivated can bedetermined using any known method, including, but not limited to,detecting production of a detectable marker operably linked to an HIVpromoter; and detecting production of a viral protein undertranscriptional control of an HIV promoter. Detection of a detectablemarker is carried out using a method suitable to the particular marker.For example, where the marker is a fluorescent protein, fluorescence isdetected; where the marker is a luminescent protein, luminescence isdetected; and the like. Such methods are well known to those skilled inthe art.

[0066] Effective amounts of exemplary reactivating agents are asfollows: from about 5 nM to about 10 nM TPA; from about 5 ng/ml to about20 ng/ml TNF-α; from about 2.5 μg/ml to about 10 μg/ml PHA; from about2.5 μg/ml to about 10 μg/ml anti-CD3 antibody; and from about 200 nM toabout 800 nM TSA. Non-limiting examples of effective amounts ofexemplary reactivating agents are as follows: 10 nM TPA; 10 ng/ml TNF-α;5 μg/ml PHA; 5 μg/ml anti-CD3 antibody; and 400 nM TSA.

[0067] Suitable periods of time for contacting a cell with areactivating agent are from about 0.5 hour to about 24 hours, e.g., fromabout 1 hour to about 2 hours, from about 2 hours to about 4 hours, fromabout 4 hours to about 8 hours, from about 8 hours to about 12 hours,from about 12 hours to about 16 hours, from about 16 hours to about 20hours, or from about 20 hours to about 24 hours. Contacting a cell withan effective amount of a reactivating agent is typically conducted understandard culture conditions of 37° C. and 5% CO₂.

[0068] The latent immunodeficiency virus (or latent immunodeficiencyvirus-based retroviral vector in the subject isolated cells istranscription competent, and includes a fully functional transactivatorprotein (Tat) and transcriptional activation response region (TAR). TheTAR, which is part of the long terminal repeat (LTR), is bound by Tatprotein during transcription activation. In many embodiments, theencoded Tat protein and the TAR are wild-type. In other embodiments, theencoded Tat protein and/or the TAR include one or more amino acid ornucleotide sequence changes compared to the wild-type, but remain fullyfunctional, e.g., Tat binds to TAR, and Tat-dependent transcriptionoccurs when the subject cell is contacted with an activating agent.

[0069] In many embodiments, the latent transcription-competentimmunodeficiency virus (or latent transcription-competentimmunodeficiency virus-based retroviral vector) is not replicationcompetent. In many of these embodiments, a portion or the entirenucleotide sequence that encodes the envelope protein in the nativeimmunodeficiency virus is deleted (e.g., not included in the vector),and the immunodeficiency virus cannot be packaged without a helper virusvector. Vectors that do not encode an immunodeficiency virus envelopeprotein are used in many embodiments because of the ease of cloning andelimination of toxicity associated with the envelope protein. In theseembodiments, the latent transcription-competent immunodeficiency virus(or latent transcription-competent immunodeficiency virus-basedretroviral vector) does not form infectious virions when reactivated.

[0070] Where the immunodeficiency virus is recombinant and is notreplication competent, viral particles are generated using a helpervirus which provides the viral proteins that are not encoded by therecombinant immunodeficiency virus. Those skilled in the art arefamiliar with helper virus constructs and packaging cell lines that areused to package viral constructs (recombinant virus) that lacks codingsequences for one or more viral proteins, e.g., gag and env, that arerequired for packaging.

[0071] In some embodiments, the latent immunodeficiency virus in thesubject cells is replication competent, e.g., the latentimmunodeficiency virus, when reactivated, is transcribed, viral proteinsare translated, and the resultant viral genome can replicate in apermissive cell. Whether the latent immunodeficiency virus isreplication competent can be determined using known methods.

[0072] In some embodiments, the latent immunodeficiency virus in thesubject cells forms infectious virions when reactivated, e.g., thelatent immunodeficiency virus, when reactivated, is transcribed, viralproteins are translated, and infectious virions are formed. Whether thelatent immunodeficiency virus forms infectious virus when reactivatedcan be determined by reactivating the latent immunodeficiency virus, asdescribed above, and determining infectivity of culture supernatant onpermissive cells. Any known method can be used. As one non-limitingexample, a subject cell containing a latent HIV is reactivated asdescribed above, and culture supernatant collected. The presence and/ornumber of infectious particles are determined by infecting Jurkat cellswith the culture supernatant.

[0073] In many embodiments, the latent immunodeficiency virus isintegrated in the genome at or near an alphoid repeat (e.g., adjacentto, or within an alphoid repeat, or within from about 10 base pairs (bp)to about 50 bp, from about 50 bp to about 100 bp, from about 100 bp toabout 500 bp, from about 500 bp to about 1 kilobase pairs (kb), fromabout 1 kb to about 5 kb, or from about 5 kb to about 10 kb of analphoid repeat). Alphoid repeats are approximately 171 base-pair repeatsand are the smallest subunit of the alpha satellite, the major componentof centromeres. Alphoid repeats are known in the art and the sequencesof numerous centromeric alphoid repeats are publicly available, e.g.,GenBank Accession Nos. AF153368; D29750; X03113; X03115; X66291; andM16101.

[0074] Cells

[0075] Any of a variety of cell can comprise a transcription-competentimmunodeficiency virus integrated into the genome of the cell. In someembodiments, the cell is an immortalized cell. In other embodiments, thecell is a primary cell culture and is not immortalized. In general, thecell is a T cell or a T cell line. In many embodiments, the cell is a Tcell or an immortalized T cell line that is permissive for animmunodeficiency virus, e.g., can be infected by an immunodeficiencyvirus, e.g., the T cell or immortalized T cell line expresses on itscell surface a CD4 receptor and a co-receptor (e.g., CXCR4 or CCR5).

[0076] Suitable immortalized T cell lines include, but are not limitedto, Jurkat; MOLT-16; MOLT-17; MOLT-3; MOLT-4, Karpas-299; HuT78; HSB-2;CCRF-CEM; SupT1; H9; and the like. Such cell lines are publiclyavailable, e.g., from the American Type Culture Collection.

[0077] Primary cultures of T cells can be obtained using standardmethods. For example, human peripheral blood mononuclear cells (PBMC)are removed from a human donor, and the T lymphocytes are separated fromother lymphoid cells by any known method, including, but not limited toFicoll-Hypaque cell separation. The cells can then be further subjectedto cell sorting on the basis of cell surface expression of CD4 and CD3molecules, e.g., using a fluorescence activated cell sorter and labeledantibody specific for CD4 and for CD3. The cells are then stimulated inthe presence of PHA and grown continuously in the presence of lowconcentrations of recombinant IL-2, according to standard protocols.

[0078] In some embodiments, a subject isolated cell is an isolatedclonal cell line. In some embodiments, a subject isolated cell is amember of a homogeneous population of cells (e.g., a population ofcloned cells from a single cloned cell line). The immunodeficiency virusneed not be integrated at the same genomic site in each cell of apopulation, and to that extent, the population can be consideredheterogeneous, even though the cells used to make the population arefrom a single cell line.

[0079] Immunodeficiency Virus and Vectors

[0080] The latent immunodeficiency virus in the subject isolated cellsis transcription competentt. The latent immunodeficiency virus can bewild-type or recombinant. In many embodiments, the latentimmunodeficiency virus is recombinant. Recombinant immunodeficiencyvirus is also referred to as “immunodeficiency virus-derived vector,”“immunodeficiency virus-based vector,” or “immunodeficiency virus-basedretroviral vector.” In many embodiments, the recombinantimmunodeficiency virus-based vector is generated using standardrecombinant DNA methods, and comprises a detectable marker for viralexpression (transcription).

[0081] In some embodiments, the latent immunodeficiency virus is awild-type immunodeficiency virus. HIV genome sequences are known in theart for a variety of HIV-1 and HIV-2 strains, and can be found inGenBank under various accession numbers, including AJ203647, AAAJ302646;AF133821, NC001802, L36874, and NC001722. SIV genome sequences are knownin the art for a variety of SIV strains, and can be found in GenBankunder various accession numbers, including AF334679, and NC001549. Anyof a variety of strains and quasispecies can be used.

[0082] In many embodiments, the transcription-competent immunodeficiencyvirus is recombinant, e.g., the immunodeficiency virus comprisesheterologous (non-immunodeficiency virus) sequences. In many of theseembodiments, the recombinant immunodeficiency virus is in a vector.Suitable vectors include, but are not limited to, plasmid vectors;Semliki forest virus vectors; vaccinia virus vectors; adenoviralvectors; and the like. Many such vectors are available commercially. Toprepare the constructs, the immunodeficiency polynucleotide is insertedinto a vector, typically by means of DNA ligase attachment to a cleavedrestriction enzyme site in the vector.

[0083] In some embodiments, the recombinant immunodeficiency viruscomprises a nucleotide sequence that encodes a detectable markerprotein. Suitable detectable marker proteins include, but are notlimited to, fluorescent proteins (e.g., a green fluorescent protein(GFP) (including enhanced GFP, e.g., available from Clontech); afluorescent protein from an Anthozoa species (as described in, e.g.,Matz et al. (1999) Nat. Biotech. 17:969-973); β-galactosidase;luciferase; and the like. The nucleotide sequence encoding thedetectable marker is operably linked to a promoter. In general, thepromoter is an immunodeficiency virus promoter, and the detectablemarker provides a read-out for transcriptional activity of theimmunodeficiency virus.

[0084] Recombinant vectors need not include the entire immunodeficiencyvirus genome. In many embodiments, a recombinant vector includes atleast the long terminal repeat (LTR) from the immunodeficiency virus, anucleotide sequence encoding the Tat protein, and a nucleotide sequenceencoding a detectable marker, where both the Tat-encoding sequence andthe detectable marker-encoding sequence are operably linked to the viralLTR, e.g., are under transcriptional control of the viral LTR.

[0085] The recombinant vector may further include other elements, suchas sequences necessary for propagation of the vector, such as an originof replication for replication in a bacterial of eukaryotic cell;sequences encoding a selectable marker for selection of bacterial cellsthat contain the vector, such as antibiotic resistance genes (e.g.,ampicillin resistance; and the like). Such elements are well known tothose skilled in the art.

[0086] In one particular embodiment, the recombinant immunodeficiencyvirus is an HIV-derived vector as described in the Examples and referredto as LTR-Tat-IRES-GFP, where both Tat and GFP coding sequences areunder transcriptional control of the HIV long terminal repeat (LTR).Both open reading frames are translated from a single mRNA due to thepresence of an internal ribosome entry site (IRES) derived from theencephalomyocarditis virus.

[0087] A number of IRES elements are known in the art, and any such IREScan be used in a recombinant immunodeficiency virus-based vector.Naturally occurring IRES sequences are known in the art and include, butare not limited to, IRES sequences derived from mengovirus, bovine viraldiarrhea virus (BVDV), hepatitis C virus (HCV; e.g., nucleotides1202-1812 of the nucleotide sequence provided under GenBank Accessionnumber AJ242654), GTX, Cyr61a, Cyr61b, poliovirus, the immunoglobulinheavy-chain-binding protein (BiP), immunoglobulin heavy chain, apicornavirus, murine encephalomyocarditis virus, poliovirus, and footand mouth disease virus (e.g., nucleotide numbers 600-1058 of thenucleotide sequence provided under GenBank Accession No. AF308157).Other IRES sequences such as those reported in WO 96/01324; WO 98/49334;WO 00/44896; and U.S. Pat. No. 6,171,821 can be used.

[0088] Methods of Generating a Cell with Latent Immunodeficiency Virus

[0089] The present invention provides a method of making an isolatedcell that comprises, integrated into the genome of the cell, atranscription-competent immunodeficiency virus (or atranscription-competent immunodeficiency virus-based vector), such thatunder basal in vitro culture conditions, the immunodeficiency virus islatent, and expression of the latent immunodeficiency virus can bereactivated. The method generally involves introducing into a populationof cells in vitro a recombinant, transcription-competentimmunodeficiency virus (or a transcription-competent immunodeficiencyvirus-based vector) that comprises a nucleotide sequence encoding aselectable marker operably linked to an immunodeficiency virus LTRpromoter; and selecting a cell from the population that comprises therecombinant immunodeficiency virus integrated into the genome of thecell, and that does not produce the detectable marker.

[0090] In many embodiments, the method involves infecting a populationof cells in vitro with a transcription-competent immunodeficiencyvirus-based vector that comprises a nucleotide sequence encoding aselectable marker operably linked to an immunodeficiency virus LTRpromoter; and selecting a cell from the population that comprises therecombinant immunodeficiency virus integrated into the genome of thecell, and that does not produce the detectable marker.

[0091] The recombinant immunodeficiency virus is introduced into cellsusing any known means, including, but not limited to, electroporation,calcium phosphate precipitation, infection (where the recombinantimmunodeficiency virus is packaged into a viral particle), and the like.

[0092] The recombinant immunodeficiency virus is contacted with the cellpopulation at a low multiplicity of infection (MOI) to reduce thelikelihood that more than one recombinant virus enters a given cell. Asuitable MOI is from about 0.01 to about 0.05, or from about 0.05 toabout 0.1.

[0093] In many embodiments, the detectable marker is a fluorescentprotein, and detection of the marker is by flow cytometry, using afluorescence activated cell sorter (FACS). In many embodiments, theselection step involves selecting a population of cells that, underbasal in vitro culture conditions, does not fluoresce or that has lowfluorescence, such that the relative fluorescence units are from 10⁰ toabout 10¹. This first selected population includes both uninfected cellsand cells that are have latent HIV.

[0094] In general, the first selected population is subjected to atleast one additional selection. A second selection is achieved bycontacting the first selected population for a suitable period of timewith an agent that reactivates the latent HIV. When the first selectedpopulation is contacted with an agent that reactivates the latent HIV, aproportion of the first selected population will exhibit no or lowlevels of detectable marker, and a proportion will exhibit higher levelsof detectable marker. In this second selection step, cells that exhibitfluorescence in a range of from about 10² to about 10³ or higher,relative fluorescence units are selected, and are a second selectedpopulation of cells. The second selected population of cells may then besubjected to a third selection step.

[0095] A third selection step involves maintaining the second selectedpopulation for a suitable period of time under basal in vitro cultureconditions, and selecting a population that exhibits no or lowfluorescence such that the relative fluorescence units are from 10⁰ toabout 10¹, which population is a third selected population.

[0096] Any of the first, second, or third selected populations issubjected to cloning, e.g., limiting dilution cloning. Cells are platedin individual wells of a multi-well plate at a density of one cell perwell.

[0097] Screening Methods

[0098] The present invention further provides screening methods foridentifying an agent that activates a latent immunodeficiency virus. Themethods generally involve contacting a subject cell that comprises atranscription-competent latent immunodeficiency virus (or atranscription-competent recombinant immunodeficiency virus-based vector)integrated into the genome of the cell with a test agent; anddetermining whether the latent immunodeficiency virus is activated.

[0099] The present invention further provides screening methods foridentifying an agent that blocks reactivation of latent immunodeficiencyvirus in response to a T cell activation signal. The methods generallyinvolve contacting a subject cell that comprises atranscription-competent latent immunodeficiency virus (or atranscription-competent recombinant immunodeficiency virus-based vector)integrated into the genome of the cell with a test agent and an agentthat activates T cells; and determining whether the latentimmunodeficiency virus is activated.

[0100] The terms “candidate agent,” “agent”, “substance,” “test agent,”and “compound” are used interchangeably herein. Candidate agentsencompass numerous chemical classes, and are generally synthetic,semi-synthetic, or naturally occurring inorganic or organic molecules.Candidate agents may be small organic compounds having a molecularweight of more than 50 and less than about 2,500 daltons. Candidateagents may comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and mayinclude at least an amine, carbonyl, hydroxyl or carboxyl group, and maycontain at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

[0101] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

[0102] A candidate agent is assessed for any cytotoxic activity it mayexhibit toward control cells not infected with an immunodeficiencyvirus, using well-known assays, such as trypan blue dye exclusion, anMTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazoliumbromide]) assay, and the like. Agents that do not exhibit cytotoxicactivity toward control cells not infected with an immunodeficiencyvirus are considered suitable candidate agents.

[0103] Assays of the invention usually include one or more controls.Thus, a test sample includes a test agent, and a control sample has allthe components of the test sample except for the test agent.

[0104] A variety of reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as nuclease inhibitors,anti-microbial agents, etc. may be used. The components may be added inany order. Incubations are performed at any suitable temperature,typically between 37° C. and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapidhigh-throughput screening. Typically between 0.1 and 1 hour will besufficient.

[0105] In some embodiments, the subject method of identifying an agentthat activates a latent human immunodeficiency virus involves contactinga subject cell with a test agent, which cell comprises a recombinant,genomically-integrated, latent, transcription-competent HIV thatcomprises a nucleotide sequence encoding a selectable marker operablylinked to an HIV promoter (as described above); and determining theeffect, if any, of the test agent on production of the detectablemarker, wherein production of the detectable marker indicates that thetest agent activates a latent HIV.

[0106] In other embodiments, the subject method for identifying an agentthat blocks reactivation of latent immunodeficiency virus in response toa T cell activation signal involves contacting a subject cell with atest agent and a reactivating agent, which subject cell comprises arecombinant genomically-integrated, latent, transcription-competent HIVthat comprises a nucleotide sequence encoding a selectable markeroperably linked to an HIV promoter; and determining whether the latentimmunodeficiency virus is activated. A decrease in production of thedetectable marker, compared to a control lacking the test agent,indicates that the test agent blocks activation of the latent HIV.Suitable reactivating agent include those described above.

[0107] Active Agents

[0108] The invention further provides an agent that activates a latentimmunodeficiency virus in a cell; and an agent that blocks reactivationof latent immunodeficiency virus. In many embodiments, the agent isidentified using a screening method of the invention.

[0109] In general, the agent is one that is biocompatible, e.g., thatdoes not exhibit any untoward effects toward cells not infected with animmunodeficiency virus, where untoward effects include cytotoxicity,induction of inflammation, induction of cell proliferation, and thelike.

[0110] In some embodiments, an active agent has a structure representedby the generic formula #1 as set forth below:

[0111] Generic Formula #1

[0112] and stereoisomers, solvates, and pharmaceutically acceptablesalts thereof, and a pharmaceutically acceptable carrier, diluent orexcipient, where:

[0113] each of R₁, R₂, and R₃ is independently selected from directbond, H, hydryoxyl, aryl, alkyl, cycloalkyl, and —NH₂; R₄ is H, —OH,alkyl, aryl and heteroaryl; A is C or N; d each of B and D isindependently CH₂ or NH.

[0114] In generic formula #1, in some embodiments, each of R₁, R₂, andR₃ is independently selected from alkyl, aryl and heteroaryl, whereineach of alkyl, aryl and heteroaryl may be substituted with one or moregroups selected from C₁-C₂₀alkyl, C₆-C₁₀aryl, heteroalkyl andheteroaryl. In some embodiments, each of R₁, R₂, and R₃ is independentlyselected —O—CH₃ and —OH.

[0115] In generic formula #1, R₄ is H, —OH, alkyl, aryl and heteroaryl.In some embodiments, R₄ is a single substituted or unsubstituted arylgroup or multiple substituted or unsubstituted aryl groups.

[0116] In other embodiments, an active agent has a structure representedby the generic formula #2 as set forth below,

[0117] Generic Formula #2

[0118] and stereoisomers, solvates, and pharmaceutically acceptablesalts thereof, and a pharmaceutically acceptable carrier, diluent orexcipient, where:

[0119] each of R₁ and R₂ is independently selected from direct bond, H,hydryoxyl, aryl, alkyl, cycloalkyl, and —NH₂; and each of A and B isindependently N or CH.

[0120] In other embodiments, an active agent has a structure representedby the generic formula #3 as set forth below,

[0121] Generic Formula #3

[0122] and stereoisomers, solvates, and pharmaceutically acceptablesalts thereof, and a pharmaceutically acceptable carrier, diluent orexcipient, where

[0123] each of R₁, R₂, R₃, R₄, and R₅ is independently selected fromdirect bond, H, hydryoxyl, aryl, alkyl, cycloalkyl, and —NH₂. In someembodiments, each of R₁, R₂, R₃, and R₄ is independently selected fromalkyl, aryl and heteroaryl, wherein each of alkyl, aryl and heteroarylmay be substituted with one or more groups selected from C₁-C₂₀alkyl,C₆-C₁₀aryl, heteroalkyl and heteroaryl.

[0124] In particular embodiments, an active agent is an agent having anyone of the structures depicted in Example 2, and stereoisomers,solvates, and pharmaceutically acceptable salts thereof, and apharmaceutically acceptable carrier, diluent or excipient.

[0125] Formulations

[0126] In general, a subject agent is prepared in a pharmaceuticallyacceptable composition for delivery to a host.

[0127] Pharmaceutically acceptable carriers preferred for use with asubject agent may include sterile aqueous of non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,and microparticles, including saline and buffered media. Parenteralvehicles include sodium chloride solution, Ringer's dextrose, dextroseand sodium chloride, lactated Ringer's or fixed oils. Intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like. Acomposition comprising a subject agent may also be lyophilized usingmeans well known in the art, for subsequent constitution and useaccording to the invention.

[0128] In general, the pharmaceutical compositions can be prepared invarious forms, such as granules, tablets, pills, suppositories,capsules, suspensions, salves, lotions and the like. Pharmaceuticalgrade organic or inorganic carriers and/or diluents suitable for oraland topical use can be used to make up compositions comprising thetherapeutically-active compounds. Diluents known to the art includeaqueous media, vegetable and animal oils and fats. Stabilizing agents,wetting and emulsifying agents, salts for varying the osmotic pressureor buffers for securing an adequate pH value, and skin penetrationenhancers can be used as auxiliary agents. Preservatives and otheradditives may also be present such as, for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like. In oneembodiment, a subject agent formulation comprises additionalanti-mycobacterial and/or anti-bacterial agent(s).

[0129] A subject agent can be administered in the absence of agents orcompounds that might facilitate uptake by target cells. A subject agentcan be administered with compounds that facilitate uptake of a subjectagent by target cells (e.g., by macrophages) or otherwise enhancetransport of a subject agent to a treatment site for action. Absorptionpromoters, detergents and chemical irritants (e.g., keratinolyticagents) can enhance transmission of a subject agent into a target tissue(e.g., through the skin). For general principles regarding absorptionpromoters and detergents which have been used with success in mucosaldelivery of organic and peptide-based drugs, see, e.g., Chien, NovelDrug Delivery Systems, Ch. 4 (Marcel Dekker, 1992). Examples of suitablenasal absorption promoters in particular are set forth at Chien, supraat Ch. 5, Tables 2 and 3; milder agents are preferred. Suitable agentsfor use in the method of this invention for mucosal/nasal delivery arealso described in Chang, et al., Nasal Drug Delivery, “Treatise onControlled Drug Delivery”, Ch. 9 and Tables 3-4B thereof, (MarcelDekker, 1992). Suitable agents which are known to enhance absorption ofdrugs through skin are described in Sloan, Use of Solubility Parametersfrom Regular Solution Theory to Describe Partitioning-Driven Processes,Ch. 5, “Prodrugs: Topical and Ocular Drug Delivery” (Marcel Dekker,1992), and at places elsewhere in the text. All of these references areincorporated herein for the sole purpose of illustrating the level ofknowledge and skill in the art concerning drug delivery techniques.

[0130] A colloidal dispersion system may be used for targeted deliveryof the subject agent to specific tissue. Colloidal dispersion systemsinclude macromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes.

[0131] Liposomes are artificial membrane vesicles which are useful asdelivery vehicles in vitro and in vivo. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 Fm canencapsulate a substantial percentage of an aqueous buffer comprisinglarge macromolecules. RNA and DNA can be encapsulated within the aqueousinterior and be delivered to cells in a biologically active form(Fraley, et al., (1981) Trends Biochem. Sci., 6:77). The composition ofthe liposome is usually a combination of phospholipids, particularlyhigh-phase-transition-temperature phospholipids, usually in combinationwith steroids, especially cholesterol. Other phospholipids or otherlipids may also be used. The physical characteristics of liposomesdepend on pH, ionic strength, and the presence of divalent cations.Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0132] Where desired, targeting of liposomes can be classified based onanatomical and mechanistic factors. Anatomical classification is basedon the level of selectivity, for example, organ-specific, cell-specific,and organelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0133] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various well known linking groups can be used forjoining the lipid chains to the targeting ligand (see, e.g., Yanagawa,et al., (1988) Nuc. Acids Symp. Ser., 19:189; Grabarek, et al., (1990)Anal. Biochem., 185:131; Staros, et al., (1986) Anal. Biochem. 156:220and Boujrad, et al., (1993) Proc. Natl. Acad. Sci. USA, 90:5728).Targeted delivery of a subject agent can also be achieved by conjugationof a subject agent to a the surface of viral and non-viral recombinantexpression vectors, to an antigen or other ligand, to a monoclonalantibody or to any molecule which has the desired binding specificity.

[0134] Utility

[0135] The subject isolated cells and the subject active agents areuseful in research, screening, and therapeutic applications. Subjectcells are useful as research tools for investigating the mechanism ofimmunodeficiency virus latency. The subject cells are also useful inscreening methods for identifying agents that reactivate latentimmunodeficiency virus, and agents that block reactivation of latentimmunodeficiency virus, which methods are described above. The subjectagents are useful in treatment methods, as described below.

[0136] Treatment Methods

[0137] The invention further provides methods of treating animmunodeficiency virus infection in an individual; methods of reducingthe reservoir of latent immunodeficiency virus in an individual. Themethods generally involve administering to an individual in need thereofan effective amount of a subject agent that activates latentimmunodeficiency virus. In many embodiments, the agent is administeredas part of a combination therapy with at least one other anti-viraltherapeutic agent.

[0138] The invention further provides methods of blocking reactivationof latent immunodeficiency virus in an individual. The methods generallyinvolve administering to an individual in need thereof an effectiveamount of a subject agent that blocks activation of latentimmunodeficiency virus. In many embodiments, the agent is administeredas part of a combination therapy with at least one other anti-viraltherapeutic agent.

[0139] An effective amount of a subject agent that reactivates latentHIV is an amount that reactivates latent HIV and reduces the reservoirof latent HIV in an individual by at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, or at least about 90%. A “reductionin the reservoir of latent HIV” (also referred to as “reservoir oflatently infected cells”) is a reduction in the number of cells in theindividual that harbor a latent HIV infection. Whether the reservoir oflatently infected cells is reduced can be determined using any knownmethod, including the method described in Blankson et al. (2000) J.Infect. Disease 182(6):1636-1642.

[0140] In general, reactivation of latent HIV in a cell results in deathof the cell. Thus, in some embodiments, an effective amount of a subjectagent that reactivates latent HIV is an amount that amount of a subjectagent that reactivates latent HIV is an amount that kills 10², 5×10²,10³, 5×10³, 10⁴, 5×10⁴, 10⁵, or more, cells in an individual, whichcells harbor latent HIV.

[0141] The amount of subject agent that is administered will vary withthe nature of the drug. As one non-limiting example, a subject agent canbe administered in the range of about 0.2 to 20 mg/kg/day. Thedetermination of how large a dosage to be used may be determined usingthe small animal model and relating the dosage based onpharmacokinetics, e.g. with equations predictive of interspeciesscaling. Usually, the lowest effective dose will be used.

[0142] An agent is administered once a day, twice daily, twice a week,or three times per week, for a period of from about 24 hours to about 7days, from about 7 days to about 2 weeks, from about 2 weeks to about 4weeks, from about 4 weeks to about 8 weeks, from about 8 weeks to about12 weeks, from about 12 weeks to about 24 weeks, or longer.

[0143] Routes of Administration

[0144] A subject agent is administered to an individual using anyavailable method and route suitable for drug delivery.

[0145] Conventional and pharmaceutically acceptable routes ofadministration include intranasal, intramuscular, intratracheal,intratumoral, subcutaneous, intradermal, topical application,intravenous, rectal, nasal, oral and other parenteral routes ofadministration. Routes of administration may be combined, if desired, oradjusted depending upon the subject agent and/or the desired effect onthe immune response. The subject agent can be administered in a singledose or in multiple doses, and may encompass administration of boosterdoses, to maintain the desired effect.

[0146] A subject agent can be administered to a host using any availableconventional methods and routes suitable for delivery of conventionaldrugs, including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes.

[0147] Inhalational routes of administration (e.g., intranasal,intrapulmonary, and the like) are particularly useful in stimulating animmune response for prevention or treatment of infections of therespiratory tract. Such means include inhalation of aerosol suspensionsor insufflation of a composition of the invention. Nebulizer devices,metered dose inhalers, and the like suitable for delivery ofcompositions to the nasal mucosa, trachea and bronchioli are well knownin the art and will therefore not be described in detail here. Forgeneral review in regard to intranasal drug delivery, see, e.g., Chien,Novel Drug Delivery Systems, Ch. 5 (Marcel Dekker, 1992).

[0148] Parenteral routes of administration other than inhalationadministration include, but are not necessarily limited to, topical,transdermal, subcutaneous, intramuscular, intraorbital, intraspinal,intrastemal, and intravenous routes, i.e., any route of administrationother than through the alimentary canal. Parenteral administration canbe carried to effect systemic or local delivery of subject agent.

[0149] Systemic administration typically involves intravenous,intradermal, subcutaneous, or intramuscular administration orsystemically absorbed topical or mucosal administration ofpharmaceutical preparations. Mucosal administration includesadministration to the respiratory tissue, e.g., by inhalation, nasaldrops, ocular drop, etc.; anal or vaginal routes of administration,e.g., by suppositories; and the like. A subject agent can also bedelivered to the subject by enteral administration. Enteral routes ofadministration include, but are not necessarily limited to, oral andrectal (e.g., using a suppository) delivery.

[0150] Any of a variety of methods can be used to determine whether atreatment method is effective. For example, methods of determiningwhether the methods of the invention are effective in treating animmunodeficiency virus infection, are any known test for indicia ofimmunodeficiency virus infection, including, but not limited to,measuring viral load, e.g., by measuring the amount of immunodeficiencyvirus in a biological sample, e.g., using a polymerase chain reaction(PCR) with primers specific for an immunodeficiency virus polynucleotidesequence; detecting and/or measuring a polypeptide encoded by animmunodeficiency virus, e.g., p24, gp120, reverse transcriptase, using,e.g., an immunological assay with an antibody specific for thepolypeptide; and measuring CD4 cell count in the individual. Methods ofassaying an immunodeficiency virus infection (or any indicia associatedwith an immunodeficiency virus infection) are known in the art, and havebeen described in numerous publications such as HIV Protocols (Methodsin Molecular Medicine, 17) N. L. Michael and J. H. Kim, eds. (1999)Humana Press.

[0151] Combination Therapies

[0152] A subject agent can be administered to an individual incombination (e.g., in the same formulation or in separate formulations)with another therapeutic agent (“combination therapy”). The subjectagent can be administered in admixture with another therapeutic agent orcan be administered in a separate formulation. When administered inseparate formulations, a subject agent and another therapeutic agent canbe administered substantially simultaneously (e.g., within about 60minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20minutes, about 10 minutes, about 5 minutes, or about 1 minute of eachother) or separated in time by about 1 hour, about 2 hours, about 4hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours,about 36 hours, or about 72 hours, or more.

[0153] Therapeutic agents that can be administered in combination withan effective amount of an agent that inhibits one or moreimmunodeficiency virus functions, which functions include, but are notlimited to, viral replication; viral protease activity; viral reversetranscriptase activity; viral entry into a cell; viral integraseactivity; activity of one or more of Rev, Tat, Nef, Vpr, Vpu, and Vif;and the like.

[0154] Therapeutic agents that can be administered in combinationtherapy, include, but are not limited to, anti-inflammatory, anti-viral,anti-fungal, anti-mycobacterial, antibiotic, amoebicidal,trichomonocidal, analgesic, anti-neoplastic, anti-hypertensives,anti-microbial and/or steroid drugs, to treat antiviral infections. Insome embodiments, patients are treated with a subject agent incombination with one or more of the following; beta-lactam antibiotics,tetracyclines, chloramphenicol, neomycin, gramicidin, bacitracin,sulfonamides, nitrofurazone, nalidixic acid, cortisone, hydrocortisone,betamethasone, dexamethasone, fluocortolone, prednisolone,triamcinolone, indomethacin, sulindac, acyclovir, amantadine,rimantadine, recombinant soluble CD4 (rsCD4), anti-receptor antibodies(e.g., for rhinoviruses), nevirapine, cidofovir (Vistide™), trisodiumphosphonoformate (Foscarnet™), famcyclovir, pencyclovir, valacyclovir,nucleic acid/replication inhibitors, interferon, zidovudine (AZT,Retrovir™), didanosine (dideoxyinosine, ddI, Videx™), stavudine (d4T,Zerit™), zalcitabine (dideoxycytosine, ddC, Hivid™), nevirapine(Viramune™), lamivudine (Epivir™, 3TC), protease inhibitors, saquinavir(Invirase™, Fortovase™), ritonavir (Norvir™), nelfinavir (Viracept™),efavirenz (Sustiva™), abacavir (Ziagen™), amprenavir (Agenerase™)indinavir (Crixivan™), ganciclovir, AzDU, delavirdine (Rescriptor™),kaletra, trizivir, rifampin, clathiromycin, erythropoietin, colonystimulating factors (G-CSF and GM-CSF), non-nucleoside reversetranscriptase inhibitors, nucleoside inhibitors, adriamycin,fluorouracil, methotrexate, asparaginase and combinations thereof.

[0155] Subjects Suitable for Treatment

[0156] The methods of the present invention are suitable for treatingindividuals who have an immunodeficiency virus infection; who are atrisk of contracting an immunodeficiency virus infection; and who weretreated for an immunodeficiency virus infection, but who relapsed. Suchindividuals include, but are not limited to, individuals with healthy,intact immune systems, but who are at risk for becoming HIV infected(“at-risk” individuals). At-risk individuals include, but are notlimited to, individuals who have a greater likelihood than the generalpopulation of becoming HIV infected. Individuals at risk for becomingHIV infected include, but are not limited to, individuals at risk forHIV infection due to sexual activity with HIV-infected individuals;intravenous drug users; individuals who may have been exposed toHIV-infected blood, blood products, or other HIV-contaminated bodyfluids; babies who are being nursed by HIV-infected mothers.

EXAMPLES

[0157] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric. Standard abbreviations may beused, e.g., h, hour(s); s or sec, second(s); min, minute(s); ml,milliliter; U, unit(s); and the like.

Example 1

[0158] Isolation, Purification, and Characterization of Cell LinesCarrying a Latent HIV

[0159] Methods

[0160] Viral Production and Cell Infections

[0161] For the production of viral particles containing the HIV-derivedvector LTR-Tat-IRES-GFP, 5×10⁶ 293T cells were transfected with plasmidspEV731 (10 μg), pCMV-R8.91 (6.5 μg), and pMD.G (3.5 μg) in 10-cm dishes.(LTR, long terminal repeat; IRES, internal ribosome entry site; GFP,green fluorescent protein). After 16 hours, the medium was replaced, andsupernatants containing viral particles were harvested 24 hours later.The number of infective particles per ml was established by infecting2×10⁵ Jurkat cells with different amounts of viral suspension. The titerof the virus stock was measured by flow cytometry analysis of GFPexpression 96 hours after infection.

[0162] To obtain a random library of clones containing the vectorLTR-Tat-IRES-GFP integrated, Jurkat cells were infected at an MOI(multiplicity of infection) of 0.1 followed by serial dilution andplating in 96-well plates. Individual clones obtained after 3 weeks wereanalyzed by flow cytometry for GFP expression. For the purification oflatently infected cells, Jurkat cells were infected with theLTR-Tat-IRES-GFP viral stock at an MOI of 0.1 and kept in culture for atleast 96 h. GFP-negative (GFP⁻) and GFP-positive (GFP⁺) cells wereseparated by fluorescence activated cell sorting (FACS) and furthercultured. One week later, GFP⁻ cells were incubated with 10 nM TPA or 10ng/ml TNF-α for 17 h.

[0163] The resulting GFP⁺ cells were sorted as a population and kept inculture or sorted into 96-well plates at 1 cell per well to generateclonal cell lines. Viral particles harboring an LTR-Tat vector wereobtained as described above by transfecting 293T cells with plasmidspEV695, pCMV-R8.91 and pMD.G, which plasmids are described in Jordan etal. (2001) EMBO J. 20:1726-1738.

[0164] Cell Culture, Transfections and Cell Treatments

[0165] Jurkat cells were grown in RPMI 1640 medium (Mediatech Cellgro,Herndon, Va.) supplemented with 10% fetal bovine serum, 100 U/ml ofpenicillin, 100 μg/ml of streptomycin, and 2 mM L-glutamine at 37° C.under a 95% air/5% CO₂ atmosphere. Peripheral blood mononuclear cells(PBMCS) were cultivated in the same medium supplemented with 100 U/mlhuman interleukin (hIL)-2 and were phytohemagglutinin (PHA)-stimulated(5 μg/ml) once every two weeks. 293T cells were grown under the sameconditions as Jurkat cells in Dulbecco's modification of Eagle's medium(Mediatech Cellgro, Herndon, Va.). 293T cells were routinely transfectedwith calcium phosphate. Jurkat cells (10⁷ cells/0.4 ml serum-freemedium) were electroporated in 0.4-cm gap cuvettes at 250 V and 950 mA(Gene Pulser II, Biorad, Hercules, Calif.). Plasmid DNA fortransfections was purified with the Qiagen Plasmid Maxi kit, followed byphenol extraction and ethanol precipitation. To test the HIV promoterinducibility, Jurkat-derived clones were incubated for 17 h with 10 nMtetradecanoyl phorbol acetate (TPA), 10 ng/ml TNF-α, 5 μg/ml PHA, 1 μMionomycin, 5 μg/ml anti-CD3 antibody, 400 nM Trichostatin A (TSA).Incubations with 5 μM aza-dC lasted for 48 h.

[0166] Flow Cytometry Analysis and Sorting

[0167] Cells were washed in PBS and resuspended in PBS containing 1%paraformaldehyde. GFP fluorescence was measured with a FACScaliburmachine (Becton Dickinson, San Jose, Calif.). A two-parameter analysisto distinguish GFP-derived fluorescence from background fluorescence wasused: GFP was measured in FL1 and cellular autofluorescence wasmonitored in FL2. Electronic compensation was applied during analysis.Analysis was gated on live cells according to forward and side scatter.A gate (R2) containing GFP-positive cells was drawn compared to anuninfected control, and the data shown refer to the percentage of cellsin R2 or mean fluorescence intensity (MFI) of those cells. Results shownthroughout the manuscript are representative three independentexperiments, except when libraries of clones were analyzed. Cell sortingwas carried out with a FACSVantage (Becton Dickinson, San Jose, Calif.).

[0168] Southern and Colony Hybridizations

[0169] Genomic DNA from infected Jurkat cells was extracted with theDNeasy Tissue kit (Qiagen, Valencia, Calif.). Southern hybridization wasperformed on digested DNA with [α-³²P]dCTP-labeled probes (MultiprimeDNA labeling system, Amersham Pharmacia Biotech, Piscataway, N.J.) asdescribed previously. Jordan et al. supra. For probes, DNA fragmentsinternal to the pEV73 1 retroviral vector were generated by PCRamplification: a 1.4-kb fragment extending between the 5′ LTR and Tatwas generated with primers EV1048 (5′-GTGGCGCCCGAACAGGGACC-3′; SEQ IDNO: 01) and EV1049 (antisense, 5′-CCGTCGAGATCCGTTCACTA-3′; SEQ ID NO:02); a 171-bp fragment corresponding to the 5′ end of the LTRs wasproduced with primers EV976 (5′-GCTAATTCACTCCCAACGAAGAC-3′; SEQ ID NO:03) and EV1333 (antisense, 5′-GCTTCTTCTACCTTCTCTTGCTC-3′; SEQ ID NO:04); a 70-bp fragments for the 3′ end of the LTRs was generated withprimers EV984 (5′-GCCCGTCTGTTGTATGACTCTG-3′; SEQ ID NO: 05) and primerEV934 (antisense, 5′-CGCCACTGCTAGAGATTTTCCAC-3′; SEQ ID NO: 06).

[0170] Alphoid PCR Amplifications and Quantitative PCR

[0171] To quantify the occurrence of integration close to alphoidrepeats, we developed a PCR assay based on previously reported methodsfor Alu elements. We used a human alpha satellite monomer consensussequence derived from 293 cloned monomers of diverse chromosomal originsto design oligonucleotides α1 (5′-AGACAGAAGCATTCTSAGAA-3′; SEQ ID NO:07), α2 (5′-ATCACAAAGNAGTTTCTSAGAAT-3′; SEQ ID NO: 08), α3(5′-TTTSATWGAGCAGNTTKGAAAC-3′; SEQ ID NO: 09) and α4(5′-AAAGAGTGTTTCMAANCTGCTCW-3′; SEQ ID NO: 10). During the first PCRreaction, genomic DNA was amplified with primer A (EV1371,5′-AGGCAAGCTTTATTGAGGCTTAAGC-3′; SEQ ID NO: 11; antisense LTR) andeither primer α1, α2, ═3, α4, Alu (EV1255,5′-TCCCAGCTACTCGGGAGGCTGAGG-3′; SEQ ID NO: 12) or B (EV1372,5′-CACACACAAGGCTACTTCCCT-3′; SEQ ID NO: 13; LTR) as a positive controlfor the presence of the LTR presence.

[0172] As negative controls, amplifications with no primer or withprimer A alone were performed. Taq DNA polymerase (0.75 U/25 μlreaction; Life Technologies, Rockville, Md.), 200 nM each dNTP, and 500nM each primer were used, and the reaction was run with the followingprogram: (a) 3 min at 94° C.; (b) 30 cycles of 30 sec at 94° C., 30 secat 53° C., and 4 min at 72° C.; and (c) 10 min at 72° C.

[0173] A second nested PCR amplification was carried out by using 1 μlof the first reaction with primers B (EV1372) and C (EV1373,5′-GCCACTCCCCIGTCCCGCCC-3′; SEQ ID NO: 14; antisense LTR), which allowsamplification of a fragment of the LTR. This second PCR was done usingsame conditions as the first one, but the extension time was 1 min andamplification was run for 25 cycles. PCR products were analyzed byethidium bromide/agarose gel electrophoresis and DNA bands werequantified with the EagleSight software (Stratagene, La Jolla, Calif.).

[0174] Real-time PCR (TaqMan) was also used to quantify integration ofHIV-derived vectors close to alphoid or Alu repeats, using amodification of a published protocol. Butler et al. (2001) Nat. Med.7:631-634. A first PCR was carried out for 25 cycles as described abovebut, instead of primer A, the LTR primer D (EV933,5′-GAGCCCTCAGATGCTGCATATAAG-3′; SEQ ID NO: 15) was used in combinationwith primers α1-4 or Alu to amplify genomic regions downstream of the3′LTR. For the nested real-time PCR, internal LTR primers E (EV1441,5′-AACTAGGGAACCCACTGCTTAAG-3′; SEQ ID NO: 16) and F (EV934,5′-CGCCACTGCTAGAGATTTTCCAC-3′; SEQ ID NO: 06; antisense) were used. Analiquot (1 μl) of the first amplification product was amplified with 200nM of each of the specific primers and 100 nM of the LTR-specific TaqManprobe G (EV1444, 5′-6 FAM-ACACTACTTGAAGCACTCAAGGCAAGCTTT-TAMRA-3′; SEQID NO:17) using the TaqMan Universal PCR Master mix (Perkin ElmerApplied Biosystems, Foster City, Calif.). The reaction was run for 40cycles (15 sec at 95° C. plus 1 min at 60° C.), in an AbiPrism 7700Sequence Detector (Perkin Elmer Applied Biosystems, Foster City,Calif.). Quantification was performed as recommended by Perkin Elmer.

[0175] Sequencing of Integrated Provirus

[0176] To check for the occurrence of mutations, the 5′ LTR fromselected clones was amplified (823-bp fragment) from genomic DNA withprimers EV976 (5′-GCTAATTCACTCCCAACGAAGAC-3′; SEQ ID NO: 03; 5′ end ofLTR) and EV987 (antisense, 5′-TCGCTTTCAGGTCCCTGTTCG-3′; SEQ ID NO: 18;gag region downstream 5′LTR). A Tat-IRES-GFP′ fragment (1,307 bp) wasamplified with primers EV1 140 (5′-CCATCGATGCCACCATGGAGCCAGTAGA-3′; SEQID NO: 19; 5′ end of Tat) and EV1253 (antisense,5′-AGGGTGTCGCCCTCGAA-3′; SEQ ID NO: 20; internal to GFP). Pfu DNApolymerase (Stratagene, La Jolla, Calif.) was used and the reaction wasrun with the following program: (a) 45 sec at 94° C.; (b) 30 cycles of45 sec at 94° C., 45 seconds at 58° C., and 1.5 minutes at 72° C.; and(c) 10 minutes at 72° C.

[0177] The amplified product was purified from ethidiumbromide-containing 1%-agarose gel with the GenClean Spin kit (Qbiogene,Carlsbad, Calif.) and cloned in the pCR-Blunt vector provided in theZero Blunt PCR Cloning kit (Invitrogen, Carlsbad, Calif.). Two to fourrecombinant clones containing the expected DNA insert were sequencedwith primers M13 Forward and M13 Reverse and the Big Dye d-RhodamineTerminator Ready Reaction kit (Perkin Elmer Applied Biosystems, FosterCity, Calif.).

[0178] HIV—Specific mRNA Measurements

[0179] RNA was isolated using TRIzol (Invitrogen) followed by digestionwith RQ1 DNase (Promega). First strand cDNA was synthesized usingSuperscript II (Invitrogen). Taqman PCR was performed on an ABIprism7700 detector using the following primer/probe set: primer 1:5′-GTGTGCCCGTCTGTTGTGTGA-3′ (SEQ ID NO: 21); primer 2:5′-GCCACTGCTAGAGATTTTCCA-3′ (SEQ ID NO: 22); probe5′-CTGGTAACTAGAGATCCC-3′ (SEQ ID NO: 23). The GAPDH primer/probe set waspurchased from Applied Biosystems.

[0180] Construction of HIV-R7/E⁻/GFP Molecular Clone

[0181] The HIV molecular clone (HIV-R7/E⁻/GFP) was constructed byintroducing a frameshift mutation in the env gene (by filling-in NdeIsite with T4 polymerase) in the backbone of HIV-R7/3/GFP. Bieniasz andCullen (2000) J. Virol. 74:9868-9877.

[0182] Sequencing of Flanking Genomic Regions

[0183] A variety of strategies were used to clone genomic DNA at theintegration site of latent clones. Inverse PCR was used to obtain thegenomic region flanking the 3′ LTR on the integrated provirus of clone#82. Briefly, genomic DNA was digested with NcoI (cleavage site betweenIRES-GFP), and the resulting products were circularized by incubatingwith T4 DNA ligase (New England Biolabs, Beverly, Mass.). A nestedseries of three inverse PCRs were performed with 3 primers for GFP(EV1253, EV1335 (5′-GGTCTTGTAGTTGCCGTCGTC-3′; SEQ ID NO: 24) and EV1336(5′-GAAGAAGATGGTGCGCTCC-3′; SEQ ID NO: 25); antisense) and 3 primers forLTR (EV933, EV996 (5′-TTGCCTGTACTGGGTCTCTCTG-3′; SEQ ID NO: 26) andEV984). Before cloning the amplification products, the presence ofLTR-containing products was confirmed by Southern hybridization with theprobe EV984/EV934 described above.

[0184] Once a particular clone was identified as containing theretroviral vector integrated close to alphoid or Alu repeats, a seriesof 2-3 nested PCR amplifications were carried out with alphoid (α1-4) orAlu (EV1255) primers and with primers for the HIV LTR (EV977(5′-ATTCCATGCAGGCTCACAGG-3′; SEQ ID NO: 27), EV1332(5′-GTGTAACAAGCGGGTGTTCTCTC-3′; SEQ ID NO: 28) and EV1333 for the 5′LTR,or EV933, EV996 and EV984 for the 3′ LTR.

[0185] To clone the integration site from clones that were not flankedby alphoid repeats, we used ligation-mediated PCR (LM-PCR) (Schmidt etal. (2001) Hum. Gene Ther. 12:743-749). Briefly, genomic DNA wasdigested with NlaIII (New England Biolabs, Beverly, Mass.) and ligatedto 100 pmoles of annealed linker cassette (oligonucleotides EV15345′-GACCCGGGAGATCTGAATTCAGTGGCACAGCAGTTAGG-3′; SEQ ID NO: 29, and EV15355′-CCTAACTGCTGTGCCACTGAATTCAG-3′; SEQ ID NO: 30). The ligation productswere used as a template in a PCR amplification with retroviral primerEV996 and linker-specific primer EV1532 (5′-GACCCGGGAGATCTGAATTC-3′; SEQID NO: 31), that amplifies the 3′ LTR and flanking genomic region. Next,an aliquot of the first PCR was used on a nested PCR with the retroviralprimer EV984 (LTR) and linker-specific primer EV1533(5′-AGTGGCACAGCAGTTAGG-3′; SEQ ID NO: 32) to increase specificity ofamplification.

[0186] In all cases, amplification products were cloned into pCR-Bluntvector and colony hybridization was used to screen for coloniesharboring an LTR-containing fragment with probe EV976/EV1333 orEV984/EV934 described above. Selected clones were sequenced asdescribed. BLAST (National Center for Biotechnology Information) wasused to compare sequences with the human genome draft sequence andnucleotide database.

[0187] Sequence of Flanking Genomic Regions in PBMCs from HIV-InfectedPatients

[0188] HIV integration site sequence in alphoid repeats. DNA wasextracted from PBMCs from HIV-infected patients completely suppressed byhighly active antiretroviral therapy.

[0189] Results

[0190] Establishment of an in vitro Model of HIV-1 Latency

[0191] To determine whether unique integration events can lead tolatency, we used an HIV-based retroviral vector containing the Tat andGFP open reading frames both under the control of the HIV promoter inthe 5′ long terminal repeat (LTR). We infected a culture of thelymphocytic cell line Jurkat with viral particles containing this vectorand used differential fluorescence-activated cell sorting (FACS) basedon GFP expression (FIG. 1A). First, we infected Jurkat cells with theLTR-Tat-IRES-GFP virus at a low MOI and isolated GFP-negative cells byFACS 4 days after infection (FIG. 1A). This population presumablyharbored both uninfected cells and cells with transcriptionally silencedproviruses. To activate HIV expression, we treated this population withTPA or tumor necrosis factor alpha (TNF-α) and purified GFP-positivecells by FACS (FIG. 1A). These cells, representing less than 0.06% ofthe original population, corresponded to the latent phenotype:GFP-negative under basal conditions and GFP-positive after activationwith TPA or TNF-α. By comparing the proportions of productively infectedcells (4%) and cells exhibiting a latent phenotype (0.06%), we calculatethat ˜1.5% of infections (1 in 66) resulted in a latent state in thissystem. These cells were both grown as a group and individually sortedfor further characterization. Reanalysis 17 days after sorting showedthat a significant proportion of the cells had no GFP expression,indicating transcriptional silencing in the absence of TPA (FIG. 1A).

[0192] Individual cells exhibiting a latent phenotype were cloned andclonal cell lines were further characterized. Flow cytometry analysis ofindividual clones 4 weeks after their isolation showed low basal GFPexpression (FIG. 1B-left panel). After TPA treatment, all clones wereactivated, and GFP levels reached a maximum that was relativelyindependent of basal GFP activity (FIG. 1B-right panel). Similar resultswere obtained after stimulation with TNF-α.

[0193] Examination of mRNA levels in these cell lines under repressed(−TNF-α) and activated conditions (+TNF-α) confirmed the presence ofvery low transcriptional levels under basal conditions and theactivation of HIV transcription by TNF-α (FIG. 1C-see clone H2).Importantly, HIV-specific transcript levels after TNF-α treatment wereof the same order of magnitude as mRNA levels measured in Jurkat cellsacutely infected with wild type HIV (NL4-3) (FIG. 1C).

[0194]FIG. 1A-C. Enrichment and purification of HIV-latently infectedcells by FACS. (A) A schematic representation of our enrichment protocolis shown. See text for details. The percentage of GFP-positive cellsobtained after infection (4%) or after TNF-α treatment of GFP- cells(0.06%) is shown. Similar data were obtained with TPA. (B) Clonal celllines isolated using the procedure described above were analyzed for GFPexpression under basal and stimulated conditions (24 hr treatment withTNF-α). (C) mRNA levels were measured for HIV and for GAPDH using TaqManPCR in untreated and TNF-α-treated cell lines. Results are expressed asa percentage of mRNA levels measured in Jurkat cells acutely infectedwith HIV_(NL4-3) (day 6 post-infection). Clone A72 is infected with anLTR-GFP construct (Jordan et al. (2001, supra), clone H2 is infectedwith the LTR-Tat-IRES-GFP vector while clones F11 and G10 are infectedwith HIV-R7/E⁻/GFP.

[0195] Mutations in a cellular factor important for HIV transcription,such as CDK9 or cyclin T1 for example, could be responsible for a latentphenotype. To test this possibility, we infected one of our latent cellline (clone 82) and control Jurkat cells with an LTR-GFP virus or withthe LTR-Tat-IRES-GFP virus (FIG. 2A). Both cell lines produced similaramounts of GFP after infection with the LTR-GFP-virus (FIG. 2A). GFPlevels were higher but also comparable in both cell lines afterinfection with the LTR-Tat-GFP virus (FIG. 2A). Similar results wereobserved in three other latent cells lines (clones H2, A2 and A10-FIG.2C).

[0196] This experiment demonstrates that the cellular environment inlatent cell lines can support HIV transcription and Tat transactivationwith the same efficiency as control Jurkat cells and is therefore notresponsible for the latent phenotype.

[0197] HIV transcription is characterized by an early, Tat-independentphase and a late, Tat-dependent phase. Since no GFP was produced by ourlatent cell lines, it is likely that Tat was not expressed since bothproteins are located on a polycistronic mRNA. To test whether Tat alonewas sufficient to reactivate latent HIV gene expression, we infectedclone 82 with a HIV-derived retroviral vector expressing the Tat protein(LTR-Tat virus) (FIG. 2B). Infection of a Jurkat clone containing asingle copy of an integrated LTR-GFP retroviral vector (clone A,described in Jordan et al. (2001) EMBO J. 20:1726-1738) with the sameLTR-Tat virus stock showed high levels of GFP expression but had noeffect in the latently infected cell line (FIG. 2B). Similar resultswere obtained after transfection of a Tat-expression plasmid into thesecell lines. Similar results were also observed in three other latentcells lines (clones H2, A2 and A10) although weak stimulation of GFPexpression was noted in clones H2 and A2 (FIG. 1C). We conclude that thelatent HIV promoter is relatively unresponsive to Tat stimulation,suggesting that the blockage of transcription in this clone liesprimarily at the level of transcription initiation.

[0198] FIGS. 2A-C. Characterization of latently infected clone. (A)Clone #82 is competent for basal and Tat-dependent HIV promoteractivity. Clone #82 was infected with viral particles containingHIV-derived vectors LTR-GFP or LTR-Tat-IRES-GFP, and GFP expression wasmeasured by flow cytometry. As a control, Jurkat cells were infected inparallel. (B) The integrated latent HIV promoter in clone #82 isunresponsive to Tat stimulation. Clone #82 was infected with aTat-expression retroviral vector, or treated with TPA, as a positivecontrol. As a control of the infection process and Tat expression, aclone containing a single integration of the HIV-derived LTR-GFP vectorwas infected in parallel. (C) HIV promoter activity (% of GFP positivecells in R2 gate) was measured in four latent clonal cell lines (82, H2,A2 and A10) 24 hr after infection with retroviral particles containingthe following vectors: LTR-GFP, LTR-Tat, LTR-Tat-GFP or after treatmentwith TNFα. Control Jurkat cells and a cell line containing a stablyintegrated LTR-GFP retroviral construct (Jordan et al. (2001) supra)were used as controls.

[0199] Preferential HIV Integration in or near Alphoid DNA in LatentlyInfected Cells

[0200] The basal activity of the HIV promoter is determined by acombination of cis- and trans-acting variables. The observationsdescribed above indicate that the cellular environment in each latentcell line is fully capable of supporting the transcription of the HIVgenome and therefore points to the role of the site of integration ofthe provirus as a likely cause for low basal transcription. Accordingly,we isolated and sequenced the provirus integration sites in 8 distinctclonal cell lines (FIG. 3A) Mapping of the integration site on the humangenome using BLAST showed that the retroviral vector had integrated inan alphoid repeat element in 4 out of 8 clones on chromosomes 7, 10 and16 (FIG. 3A). The site of integration of 3 clones was identified in thehuman genome in non-alphoid repeat DNA while the site of integration inone clone did not yield significant homology to any region of the humangenome (FIG. 3A).

[0201] To confirm this observation, we developed a novel PCR assay inwhich degenerate primers matching alphoid DNA consensus sequence (α1,α2, α3, α4) are used in combination with an LTR specific primer (primerA)(FIG. 3A). Amplified products were detected with two nested primers inthe LTR (primers B and C) (FIG. 3B). Since primers α1 and α3 areorientated in the same way in the alphoid repeat, a positive signal withone primer should be accompanied by a positive signal for the other(FIG. 3B). The same holds true for primers α2 and α4 (FIG. 3B).Similarly, a positive response with primers α1 and α3 would beaccompanied by a negative signal for primers α2 and α4. Examination ofthe PCR reactions showed a perfect concordance with the sequencing data:clones 82, A7, H2 and F2 showed a positive PCR reaction and had adocumented alphoid repeat integration while clones A1, A5, A10 and A11tested negative in the PCR assay and did not integrate in an alphoidelement.

[0202] For comparison, we subjected DNA obtained from a library of 34random integrations (not sorted for a latent phenotype) to the sameprocedure (Jordan et al. (2001), supra). None of these 34 clones showeda pattern consistent with alphoid repeat integration, in agreement withprevious reports that HIV integration in or near alphoid repeat elementsis disfavored (Carteau et al. (1998) J. Virol. 72:4005-4014).

[0203] To examine the relative distribution of alphoid integration inlatently vs. productively infected cells at a population level, weadapted the alphoid PCR assay to TaqMan PCR. Because Alu elements arerandomly distributed within the genome, this analysis included a controlusing primers specific for the Alu element and the HIV LTR. The productamplified by this primer pair is assumed to represent random integrationevents within the genome and has been used as a reliable marker of HIVintegration (Butler et al. (2001) Nat. Med. 7:631-634; Chun et al.(1997) Proc. Natl. Acad. Sci. USA 94:13193-13197; and Minami et al.(1995) Genomics 29:403-408. Results are therefore presented as the ratioof the alphoid-LTR product to the Alu-LTR product as an indication ofthe frequency of integration in or near alphoid DNA (FIG. 3D).

[0204] When cell populations corresponding to productively infectedcells (GFP positive in first panel of FIG. 1A) were compared tolatently-infected cell population (GFP positive in third panel of FIG.1A), we observed preferential integration (9-fold to >100-foldenrichment) in or near alphoid repeats in latent cells in comparison toproductively infected cells (FIG. 3D). Similar results were obtainedafter infection of human peripheral blood mononuclear cells (PBMCs) withthe HIV-derived vector. Cloning and sequencing of several PCR productsobtained after amplification with the alphoid specific primers confirmedthat these contained HIV integration events into alphoid DNA.

[0205]FIG. 3 Latency is associated with preferred integration in or nearalphoid repeats. (A) The sequence of integration site of the HIV vectoris shown for 7 clonal cell lines aligned with the corresponding genomicsequence. The match in Genbank for each clone corresponded to Genbankaccession number M93288 for clone 82, AL591625 for clone A1, AC023948for clone A5, M16037 for clone A7, A1354920 for clone A10, AC019063 forclone H2 and AC079801 for clone F2. The sequence corresponding to theHIV promoter is indicated by a closed box. The chromosomal location ofeach integration site is shown and integration sites into alphoid repeatelements are indicated. (B) A nested PCR assay designed to quantifyintegration in or near alphoid repeats is schematically represented. Theposition of primers in the two sequential PCR reactions are show alignedwith the HIV 5′ LTR and a putative alphoid element in the genome. (C)Real-time PCR analysis of integration in or near alphoid repeats. PrimerG represents an internal fluorescent primer used for the quantificationof the TaqMan reaction. (D) Quantification of PCR products after TaqManPCR analysis as indicated under D. Productively infected cells(GFP-positive cells in panel 1, FIG. 1A) (productive infection) arecompared with latently infected cells (panel 4, FIG. 1A)(latentinfection) using the Alu and alphoid PCR assay. Data is expressed as therelative signal intensity (alphoid/Alu). Numbers on top of bar indicatesthe ratio between latent and productive infection.

[0206] Transcriptional Activation of the HIV Promoter in LatentlyInfected Cells

[0207] We have tested a number of biological and chemical agents fortheir ability to reactivate latent HIV expression. Several NF-κBactivators, including TPA, TNF-α, and PHA, independently induced HIVexpression, as expected since NF-κB strongly induces the HIV promoteractivity. TPA was the strongest inducer tested (FIG. 4). All treatmentsincreased the number of GFP-positive cells and the mean fluorescenceintensity reflective of GFP levels. Incubation with anti-CD3 antibodies,which crosslink the surface T-cell receptors, an alternate pathwayleading to NF-κB activation, also induced GFP expression. These resultssuggest that activation of the NF-κB pathway may boost HIV transcriptioninitiation, the production of Tat, and transition to Tat-dependenttranscriptional activation. Trichostatin A (TSA), an inhibitor ofhistone deacetylases also activated expression but to a lesser extentthan NF-κB activators and in only some cell lines (see clones Al as anexample, FIG. 4A). Treatment with 5-axa-2-deoxycytidine (aza-dC), aninhibitor of DNA methylation, had little effect on the fraction of cellsinduced to transcribe HIV alone or in combination with a histonedeacetylase inhibitor.

[0208]FIG. 4 Transcriptional activation of the HIV promoter in latentlyinfected cells. Cells from clones 82, A1, A7 and A10 were treated asdescribed in Methods with several indicated agents and LTR expressionwas measured by flow cytometry. Data are expressed as percentage ofcells becoming GFP-positive after a 24 hr treatment.

[0209] Latent Cell Lines Containing a Full-Length Integrated HIV Genome

[0210] To confirm that HIV latency can be established in the context ofa full length provirus, we used a recombinant HIV molecular clonecontaining the GFP open reading frame in place of the Nef gene (Bieniaszand Cullen (2000) J. Virol. 74:9868-9877) (HIV-R7/E⁻/GFP)(FIG. 5A). Torestrict our analysis to a single infection cycle, the env gene wassuppressed by introduction of a frameshift mutation. This defect wascomplemented by coexpression of a VSV-G envelope protein to generatepseudotyped viral particles. We infected a culture of the lymphocyticcell line Jurkat with viral particles containing this HIV genome andused differential fluorescence-activated cell sorting based on GFPexpression to isolate GFP-negative cells by FACS 4 days after infection(FIG. 5B). Based on our previous experiments, we predicted that thispopulation harbored both uninfected cells and cells withtranscriptionally silenced proviruses. To activate HIV expression, wetreated this population with TNF-α and observed that a small fraction ofthe cell population (1.9%) became positive. These activated cells werepurified by FACS based on GFP expression levels (FIG. 5B). These cellswere both grown as a group and individually sorted for furthercharacterization.

[0211] Reanalysis after sorting showed that a small proportion of thecells had no GFP expression, indicating transcriptional silencing hasoccurred after withdrawal of TNF-α (FIG. 5B). Flow cytometry analysis ofindividual clones showed low basal GFP expression (FIG. 5C). After TNF-αtreatment, HIV expression was increased in all clones both in terms ofthe fraction of cells that became GFP positive (FIG. 5C) but also interms of mean fluorescence intensity. Measurement of virus-specific mRNAshowed that the mechanism of latency in these clones was controlled atthe transcriptional level (FIG. 1C, clones F11 and G10). Levels of viralmRNA after activation were similar to those measured after a productiveHIV infection (compare clones F11 and G10 with NL4-3, FIG. 1C). Analysisof HIV-specific protein expression in several clones by western blottingusing an antiserum from an HIV-infected individual showed no detectableexpression of HIV proteins under basal conditions. Treatment of the sameclones with TNF-α led to a dramatic increase in HIV protein expression,particularly the gag p55 precursor (FIG. 5D).

[0212]FIG. 5. Establishment of latently infected cell lines with afull-length HIV provirus. (A) genome organization of a molecular cloneof HIV encoding GFP and containing a frameshift mutation in env. (B)Schematic representation of protocol for enrichment of latently-infectedcells after infection of Jurkat cells with HIV-R7/E⁻/GFP (see text fordetail). (C) Clonal cell lines isolated using the procedure describedabove were analyzed for GFP expression under basal and stimulatedconditions (24 hr treatment with TNF-α).

[0213] When the culture supernatants of the same clones were examinedfor HIV-specific p24 expression, no or low picogram amounts could bedetected under basal conditions (Table I). Treatment with TNF-α led to agreater than a thousand-fold increase in p24 measurement for severalrepresentative clones (Table I). These observations demonstrate thattranscriptional latency can also be established in the context of afull-length HIV infection. TABLE I GFP-Positive (%) GFP Signal (MFI) p24(pg/ml) TNF-α − + − + − + Clone <1 46 ± 6 ± 0.5 188 ± 34  7 ± 12 6,066 ±1,960 15.4 6 Clone <1 27 ± 5 ± 0.2 135 ± 43 0 10,100 ± 4,573   6.3 9Clone <1 77 ± 5 ± 0.3 488 ± 74 0 32,967 ± 10,537  8.4 6 Clone <1 75 ± 7± 0.5 522 ± 61 23 ± 6  41,067 ± 9,100   9.2 7 Clone <1 96 ± 5 ± 1.4 645± 45 14 ± 3  85,500 ± 5,981  10.6 1

[0214] We were able to amplify the integration site of provirusintegrated into alphoid repeats from PBMCs from HIV-1-infectedindividuals treated with highly active antiretroviral therapy (data inFIG. 6).

Example 2

[0215] Identification of Agents that Reactivate Latent HIV

[0216] We screened a library of 6,000 small molecules (library purchasedfrom Chembridge, San Diego) using one of the cell lines containing alatent HIV-based retroviral vector. Viral expression was monitored byGFP expression, as described above. Two compounds were identified thatreproducibly lead to an induction of HIV expression as measured byincreased GFP expression. The results of a typical induction experimentin which we measured GFP expression in response to differentconcentrations of the two small molecules (Hit 1 and Hit2) are shown inFIG. 7. The structures of three representative active agents are shownbelow.

[0217] Hit #1-ID 766456-MW 366.3797-C₁₉H₁₈N₄O₄

[0218] Hit #2-ID 681306-MW 294.3592-C₁₇H₁₈N₄O

[0219] Hit #3-ID 671859-MW 317.3447-C₁₇H₁₉NO₅

[0220] From the data presented above, it is evident that the instantinvention provides isolated cells that harbor a latent immunodeficiencyvirus that is transcription competent, that can be reactivated, and thatis an in vitro model for latent HIV infection in vivo. The cells areuseful for investigating the nature of latency, and also in drugscreening assays to identify agents that activate latent HIV.Identification of agents that activate latent HIV is important, as suchagents are useful, particularly in conjunction with standard anti-HIVtherapies, to reduce the reservoir of latent HIV.

[0221] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1 50 1 20 DNA Artificial Sequence primer 1 gtggcgcccg aacagggacc 20 2 20DNA Artificial Sequence primer 2 ccgtcgagat ccgttcacta 20 3 23 DNAArtificial Sequence primer 3 gctaattcac tcccaacgaa gac 23 4 23 DNAArtificial Sequence primer 4 gcttcttcta ccttctcttg ctc 23 5 22 DNAArtificial Sequence primer 5 gcccgtctgt tgtatgactc tg 22 6 23 DNAArtificial Sequence primer 6 cgccactgct agagattttc cac 23 7 20 DNAArtificial Sequence primer 7 agacagaagc attctsagaa 20 8 23 DNAArtificial Sequence primer 8 atcacaaagn agtttctsag aat 23 9 22 DNAArtificial Sequence primer 9 tttsatwgag cagnttkgaa ac 22 10 23 DNAArtificial Sequence primer 10 aaagagtgtt tcmaanctgc tcw 23 11 25 DNAArtificial Sequence primer 11 aggcaagctt tattgaggct taagc 25 12 24 DNAArtificial Sequence primer 12 tcccagctac tcgggaggct gagg 24 13 21 DNAArtificial Sequence primer 13 cacacacaag gctacttccc t 21 14 20 DNAArtificial Sequence primer 14 gccactcccc ngtcccgccc 20 15 24 DNAArtificial Sequence primer 15 gagccctcag atgctgcata taag 24 16 23 DNAArtificial Sequence primer 16 aactagggaa cccactgctt aag 23 17 30 DNAArtificial Sequence probe 17 ncactacttg aagcactcaa ggcaagcttn 30 18 21DNA Artificial Sequence primer 18 tcgctttcag gtccctgttc g 21 19 28 DNAArtificial Sequence primer 19 ccatcgatgc caccatggag ccagtaga 28 20 17DNA Artificial Sequence primer 20 agggtgtcgc cctcgaa 17 21 21 DNAArtificial Sequence primer 21 gtgtgcccgt ctgttgtgtg a 21 22 21 DNAArtificial Sequence primer 22 gccactgcta gagattttcc a 21 23 18 DNAArtificial Sequence probe 23 ctggtaacta gagatccc 18 24 21 DNA ArtificialSequence primer 24 ggtcttgtag ttgccgtcgt c 21 25 19 DNA ArtificialSequence primer 25 gaagaagatg gtgcgctcc 19 26 22 DNA Artificial Sequenceprimer 26 ttgcctgtac tgggtctctc tg 22 27 20 DNA Artificial Sequenceprimer 27 attccatgca ggctcacagg 20 28 23 DNA Artificial Sequence primer28 gtgtaacaag cgggtgttct ctc 23 29 38 DNA Artificial Sequence linker 29gacccgggag atctgaattc agtggcacag cagttagg 38 30 26 DNA ArtificialSequence linker 30 cctaactgct gtgccactga attcag 26 31 20 DNA ArtificialSequence primer 31 gacccgggag atctgaattc 20 32 18 DNA ArtificialSequence primer 32 agtggcacag cagttagg 18 33 69 DNA Homo sapiens 33atgtgtgcgt tcaactcaca gagtttaacc tttcttttcg tagagcagtg gaagggctaa 60ttcactccc 69 34 48 DNA Homo sapiens 34 atgtgtgcgt tcaactcaca gagtttaacctttcttttcg tagagcag 48 35 69 DNA Homo sapiens 35 agctcctgac tatgataaagtatcttgtga aaaaccaatg ttactgcttg gaagggctaa 60 ttcactccc 69 36 48 DNAHomo sapiens 36 agctcctgac tatgataaag tatcttgtga aaaaccaatg ttactgct 4837 69 DNA Homo sapiens 37 cggctcactg cagcctccgc ctctcgaatt caattctgtctcagcctctg gaagggctaa 60 ttcactccc 69 38 48 DNA Homo sapiens 38cggctcactg cagcctccgc ctctcgaatt caattctgtc tcagcctc 48 39 69 DNA Homosapiens 39 ctgtgatttg aatgcacaca tcacaaagaa gtttctcaga atgcttcttggaagggctaa 60 ttcactccc 69 40 48 DNA Homo sapiens 40 ctgtgatttgaatgcacaca tcacaaagaa gtttctcaga atgcttct 48 41 69 DNA Homo sapiens 41gagttgacaa aggtaaaaca gattttttaa aaatcagttg tttatatttg gaagggctaa 60ttcactccc 69 42 48 DNA Homo sapiens 42 gagttgacaa aggtaaaaca gattttttaaaaatcagttg tttatatt 48 43 69 DNA Homo sapiens 43 ctgtgatttg aatgcacacatcacaaagga gtttctgaga atgcttcttg gaagggctaa 60 ttcactccc 69 44 48 DNAHomo sapiens 44 ctgtgatttg aatgcacaca tcacaaagga gtttctgaga atgcttct 4845 69 DNA Homo sapiens 45 ttcgtaggag aactagacag aatgattctc agaaactactttgtgatgtg gaagggctaa 60 ttcactccc 69 46 48 DNA Homo sapiens 46ttcgtaggag aactagacag aatgattctc agaaactact ttgtgatg 48 47 205 DNA Homosapiens 47 aaagagtgtt tccaagctgc tctgtcaaaa ggaaggttct tctctgttaggtgagtgcat 60 acgtcataaa ggagtttctg agaattcttc tgtctagttc ttatttgtagacgtttcctt 120 tctcacctta ggcctgaaag cgctcgaaat atccacttcc agatagtacagaaatagtga 180 ttcaaacctg gaagggctaa ttcac 205 48 230 DNA Homo sapiensmisc_feature 84 n = A,T,C or G 48 gaccctttta gtcagtgtgg aaaatctctagcaaaagggt gtttcaaacc tgctctatga 60 aagggaatgt tcaactctgt gacnttgaatgcaaatatca caaagaagtt actgggaatg 120 ctgctgtctg ctttttatat gtaatcccgtttccaacgaa atcctcaaag ctagacaaat 180 atccacttgc agattccaca aaaagagtgtttcaaatctg ctcaatcaaa 230 49 570 DNA Homo sapiens 49 tctggtccctggccctggtg tgtagttctg ccaatcaggg aagtagcctt gtgtgtggta 60 gacccacagatcaagaatat cttgtctgtt ctgggagtga actagccctt ccacctgcat 120 gtggaaattttgagcgcttt gaggcctatt gtggaaaagg aaatatgttc acataaaagc 180 tacacagaagcattctgaaa aacgtctttg tgatgagtgc attcatctca cagagttgat 240 cctttctttttattcagcag ttttgaaaca ctccttttag agaatctgca agtagatatt 300 tggagcgcgttgaggcctac catggaaaag caaatatctt cacataaaaa ctacacagaa 360 atattctcagaaactacttt gtgatatgtg tgttcaattc acagagttga acctttcttt 420 tcattgagcagttttgaaaa actgcttttc tagaatctgc ttgtggatat ttggagctct 480 ttgaggaattcattgtcaat gggatatctt catatacaaa ctagccagaa gcattctcag 540 aaactactttgtgatcctga attccagcac 570 50 569 DNA Homo sapiens misc_feature 535, 544n = A,T,C or G 50 agtccctggc cctggtgtgt agttctgcca atcagggaag tagccttgtgtgtggtagac 60 ccacagatca agaatatctt gtcttttctg ggagtaaatt agcccttccagatattaact 120 tttcaaacat aggccgcaca gtgatccaaa tatgtacctt acagatacttcaaaaagact 180 gtttccaacc tgctcaatga aaagaaagtt tcaactgtgt gagtggaatgcaaacatcac 240 aaagaagttt ctcagaatgc ccctgtcaac tttatatgtg aagatattgccttttccaca 300 aaacgcctca aaccattcca aatatccatt tgcagattcc acaaaaagactgtttccaaa 360 ctgctcaacc aaaaaaaggt tcaactctgt gagatgaatg cacccatcacaaagaagttt 420 ttcagaaagc ttctgtttag tttttatgtg aagatatttc ctttttcactataggcttca 480 aagcactcca cacatccatt tgcagattct acaaaagagt gtttccaatctgctncctga 540 attncagcac actggcggcc gttactagt 569

1. An isolated cell that comprises, integrated into the genome of thecell, a recombinant transcription-competent immunodeficiency virus-basedvector, wherein, under basal in vitro culture conditions, theimmunodeficiency virus is latent, and wherein expression of the latentimmunodeficiency virus can be reactivated.
 2. The cell of claim 1,wherein said cell is an immortalized cell line.
 3. The cell of claim 1,wherein said cell is a T lymphoid cell.
 4. The cell of claim 1, whereinsaid immunodeficiency virus is human immunodeficiency virus (HIV). 5.The cell of claim 4, wherein said immunodeficiency virus is HIV-1.
 6. Amethod of making an immortalized cell that comprises, integrated intothe genome of the cell, a recombinant, transcription-competent humanimmunodeficiency virus (HIV) vector, wherein, under basal in vitroculture conditions, the HIV is latent, and wherein expression of thelatent HIV can be reactivated, the method comprising: a) introducinginto population of immortalized cells in vitro a recombinant,transcription-competent HIV that comprises a nucleotide sequenceencoding a selectable marker operably linked to a promoter; and b)selecting a cell population that comprises the recombinant HIVintegrated into the genome of the cell, and that does not produce thedetectable marker.
 7. The method of claim 6, further comprising cloninga cell from the selected cell population.
 8. The method of claim 6,wherein step (b) results in a first selected cell population, and themethod further comprises the steps of: c) contacting said first selectedcell population with an agent that activates HIV transcription; d)selecting a second population of cells from the first selectedpopulation, which second selected population produces the selectablemarker.
 9. The method of claim 8, wherein said agent is selected fromthe group consisting of an activator of NF-κB, an agent that cross-linkscell-surface T-cell receptor, and an inhibitor of hi stone deacetylase.10. The method of claim 9, wherein said activating agent is selectedfrom the group consisting of phytohemagglutinin, tetradecanoyl phorbolacetate, TNFα, an anti-CD3 antibody, and trichostatin A.
 11. An isolatedimmortalized cell that comprises, integrated into the genome of thecell, a recombinant transcription-competent human immunodeficiency virus(HIV) vector that comprises a nucleotide sequence encoding a selectablemarker operably linked to a promoter, wherein, under basal in vitroculture conditions, the HIV is latent, and wherein expression of thelatent HIV can be reactivated.
 12. A method of identifying an agent thatactivates a latent human immunodeficiency virus (HIV), the methodcomprising: a) contacting the cell according to claim 11 with a testagent; and b) determining the effect, if any, of the test agent onproduction of the detectable marker, wherein production of thedetectable marker indicates that the test agent activates a latent HIV.13. The method of claim 12, wherein said detectable marker is afluorescent protein, and said determining is detection of fluorescence.14. A composition comprising an agent identified by the method of claim12; and a pharmaceutically acceptable excipient.
 15. A method ofreducing the number of cells containing a latent human immunodeficiencyvirus in an individual, the method comprising: administering to theindividual an effective amount of the composition of claim
 14. 16. Amethod of treating a human immunodeficiency virus infection in anindividual, the method comprising: administering to an individual aneffective amount of the composition of claim 14; and administering tothe individual an effective amount of an agent that inhibits animmunodeficiency virus function selected from the group consisting ofviral replication, viral protease activity, viral reverse transcriptaseactivity, viral entry into a cell, viral integrase activity, viral Revactivity, viral Tat activity, viral Nef activity, viral Vpr activity,viral Vpu activity, and viral Vif activity.